A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons

Florez-Paz D, Bali KK, Kuner R, et al. Scientific reports 2016;6:25923.

The test records were read three times by AI and checked by code. No person has reviewed them yet. How records are made

Balance beam test

Setting	Value	Paper’s words
Beam size	1 m long, 2 cm wide	“Balance was tested on a 1 m long, 2 cm wide beam”
Beam height	50 cm	“beam suspended on two poles 50 cm above a table top”
Number of trials	Unclear at least three times One passage covers all tests at once. It gives a lower bound on repetitions per animal but does not say whether these fell within one session or day or across sessions.	“each of which was repeated at least three times in each animal”
Other settings in the paper
Aversive stimulus	a lamp above the start point	“a lamp above the start point served as an aversive stimulus”
Box at the finish point	Food	“Food was placed in a box at the finish point to attract the mouse”
Apparatus
Balance beam		“Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top. Food was placed in a box at the finish point to attract the mouse and a lamp above the start point served as an aversive stimulus. A video camera on a tripod was used to record the time required to cross the beam.”

Gait analysis

Compare with 8 other papers

Setting	Value	Paper’s words
Walkway size	85 cm long and 8.5 cm wide	“the mice were placed in a glass chamber (85 cm long and 8.5 cm wide) and they were allowed to move freely across the walkway”
Number of runs	at least three times	“scored all the tests, each of which was repeated at least three times in each animal” “the values from the individual tracks were averaged”
Not in the Methods	Run duration criterion
Apparatus
CatWalk system 7.1	Noldus Information Technology	“The animal’s gait was tested with the CatWalk system 7.1 (Noldus Information Technology), where the mice were placed in a glass chamber (85 cm long and 8.5 cm wide) and they were allowed to move freely across the walkway.”
CatWalk software		“allowing the paw prints to be captured by a video camera and analyzed with the CatWalk software”

Rotarod test

Compare with 9 other papers

Setting	Value	Paper’s words
Rod speed	5 rpm and it accelerated to 20 rpm	“The initial speed of rotation was 5 rpm and it accelerated to 20 rpm”
Number of trials	Unclear at least three times One passage gives one repetition count for all tests, including the rotarod, as repetitions per animal. It does not say whether these were trials within one session or day or repeats across sessions, and it is only a lower bound.	“all the tests, each of which was repeated at least three times in each animal”
Not in the Methods	Trial duration
Other settings in the paper
Diameter rotating cylinder	5 cm	“the mice were placed on a 5 cm diameter rotating cylinder”
Apparatus
Rotarod	Ugo Basile	“In the rotarod test (Ugo Basile), the mice were placed on a 5 cm diameter rotating cylinder along which they must walk in order not to fall off. The initial speed of rotation was 5 rpm and it accelerated to 20 rpm.”

Facts checked by a reviewer

A person went through this paper’s whole Methods section, beyond the behavioral tests, and confirmed these 118 facts against the text. No person has checked the test records above.

Study design (1)

Item	Value	Paper’s words
This studyBlinding	The same investigator scored all the tests	“The same investigator scored all the tests”

Subjects and groups (13)

Item	Value	Paper’s words
Experiment subjectsSpecies	mouse	“Adult C57Bl/6J mice were used in this study.”
Experiment subjectsStrain	C57Bl/6J	“Adult C57Bl/6J mice were used in this study.”
Experiment subjectsAge	6–9 week-old	“6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice”
Experiment subjectsSex	male	“Male animals were used for in vitro recordings”
Experimental miceAge	6–9 week-old	“we tested a cohort of 6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice”
Pvalb-Cre:RCE:Piezo2cKO miceLabel	Pvalb-Cre:RCE:Piezo2cKO mice	“we tested a cohort of 6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice (n = 7)”
Pvalb-Cre:RCE:Piezo2cKO miceN allocated	7	“Pvalb-Cre:RCE:Piezo2cKO mice (n = 7)”
Behavioral test subjectsAge	6–9 week-old	“a cohort of 6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice (n = 7) and their control littermates (n = 7)”
Behavioral test subjectsSex	female	“6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice”
Pvalb-Cre:RCE:Piezo2cKO groupLabel	Pvalb-Cre:RCE:Piezo2cKO	“6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice (n = 7)”
Pvalb-Cre:RCE:Piezo2cKO groupN allocated	7	“6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice (n = 7)”
control littermates groupLabel	control littermates	“and their control littermates (n = 7)”
control littermates groupN allocated	7	“and their control littermates (n = 7)”

Materials and equipment (88)

Item	Value	Paper’s words
vibrating slicerName	vibrating slicer	“the brainstem was attached at its rostral end to the platform of the vibrating slicer”
vibrating slicerModel	series 1000	“the vibrating slicer (Pelco, TPI, series 1000, St. Louis, USA)”
isofluraneName	isoflurane	“animals were anesthetized by isoflurane inhalation”
DiI tracerManufacturer	Life Technology, Carlbad, USA	“the DiI tracer (5%: Life Technology, Carlbad, USA)”
DiI tracerName	DiI tracer	“the DiI tracer (5%: Life Technology, Carlbad, USA) was injected”
cyanoacrylateName	cyanoacrylate	“The incision was closed with a drop of cyanoacrylate”
artificial cerebrospinal fluid (ACSF)Name	artificial cerebrospinal fluid (ACSF)	“immersed in ice-cold, oxygenated (95% O2–5% CO2) artificial cerebrospinal fluid (ACSF)”
ACSFName	artificial cerebrospinal fluid (ACSF)	“artificial cerebrospinal fluid (ACSF)”
Multiclamp 700 amplifierName	Multiclamp 700 amplifier	“using a Multiclamp 700 amplifier”
pCLAMP 10Name	pCLAMP 10	“data were acquired using pCLAMP 10 software”
pCLAMP 10Version	10	“the data were acquired using pCLAMP 10 software”
Digidata 1440Manufacturer	Molecular Devices, Sunnyvale, USA	“Digidata 1440 (Molecular Devices, Sunnyvale, USA)”
Digidata 1440Name	Digidata 1440	“and a Digidata 1440 (Molecular Devices, Sunnyvale, USA)”
borosilicate glass capillariesName	borosilicate glass capillaries	“Patch electrodes of 4–7 MΩ were pulled from borosilicate glass capillaries”
patch electrodesName	patch electrodes	“Patch electrodes of 4–7 MΩ were pulled from borosilicate glass capillaries”
internal solutionName	internal solution	“The pipettes were filled with an internal solution containing (in mM): 115 K-gluconate, 25 KCl, 9 NaCl, 10 HEPES, 0.2 EGTA, 1 MgCl2, 3 K2-ATP and 1 Na-GTP, pH 7.2 adjusted with KOH and 280–290 mosmol Kg−1.”
external control solutionName	external control solution	“The control external solution consisted of (in mM): 140 NaCl, 3 KCl, 1 CaCl2 2 MgCl2, 10 glucose and 10 HEPES (pH 7.2).”
recording chamberName	recording chamber	“Slices were transferred to a recording chamber and perfused with oxygenated external control solution.”
fluorescence microscopeName	fluorescence microscopy (Olympus BX50WI)	“fluorescence microscopy (Olympus BX50WI)”
fluorescence microscopeManufacturer	Olympus	“fluorescence microscopy (Olympus BX50WI)”
Imago cameraManufacturer	Till photonics GmbH, Germany	“an Imago camera (both Till photonics GmbH, Germany)”
Imago cameraName	Imago camera	“an Imago camera (both Till photonics GmbH, Germany)”
Imaging Workbench 6.0Name	Imaging Workbench 6.0	“the Imaging Workbench 6.0 software (INDEC BioSystems)”
Imaging Workbench 6.0Manufacturer	INDEC BioSystems	“the Imaging Workbench 6.0 software (INDEC BioSystems)”
Imaging Workbench 6.0Version	6.0	“the Imaging Workbench 6.0 software (INDEC BioSystems)”
heat-closed glass pipetteName	heat-closed glass pipette	“using a heat-closed glass pipette driven by a MM3A-LS piezoelectric micromanipulator”
MM3A-LS piezoelectric micromanipulatorManufacturer	Kleindiek, Reutlingen, Germany	“MM3A-LS piezoelectric micromanipulator (Kleindiek, Reutlingen, Germany)”
MM3A-LS piezoelectric micromanipulatorName	MM3A-LS piezoelectric micromanipulator	“MM3A-LS piezoelectric micromanipulator (Kleindiek, Reutlingen, Germany)”
Piezo2 shRNACatalog number	TR509519	“Cat. No. TR509519: Origene, USA”
Piezo2 shRNAManufacturer	Origene, USA	“Cat. No. TR509519: Origene, USA”
AAV backbone PAM vectorName	AAV backbone of the PAM vector	“adeno-associated viral (AAV) backbone of the PAM vector”
U6 promoterName	U6 promoter	“along with the U6 promoter”
GFP reporter cassetteName	GFP reporter cassette	“The expression of the native GFP reporter cassette in the final clones was confirmed by the strong GFP signal following transfection into HEK-293 cells”
HEK-293 cellsName	HEK-293 cells	“following transfection into HEK-293 cells”
rAAV serotype 2/8 particlesName	rAAV serotype 2/8 particles	“The rAAV serotype 2/8 particles carrying shRNA against Piezo2 (AAV-Piezo2-sh-1) or a scrambled shRNA were generated in house by co-transfecting HEK-293 cells with the cloned rAAV backbone plasmid and the helper 2/8 plasmids”
AAV-Piezo2-sh-1Name	AAV-Piezo2-sh-1	“rAAV serotype 2/8 particles carrying shRNA against Piezo2 (AAV-Piezo2-sh-1)”
scrambled shRNA virusName	scrambled shRNA	“or a scrambled shRNA were generated”
Nanoliter2010Manufacturer	WPI	“nanoliter injector microprocessor (Nanoliter2010, WPI)”
glass micropipetteName	glass micropipette	“The virus was delivered using a glass micropipette”
nanoliter injector microprocessorName	Nanoliter2010	“nanoliter injector microprocessor (Nanoliter2010, WPI)”
Nanoliter2010 injectorManufacturer	WPI	“nanoliter injector microprocessor (Nanoliter2010, WPI)”
AAV-Piezo2-sh-1 injectionVolume	700 nl	“the viral solution was injected in a total volume of 700 nl”
bonewaxName	bonewax	“a layer of adhesive cement (bonewax) was applied around the hole”
cyanocrylateName	cyanocrylate	“We then sutured the skin with a tissue adhesive (cyanocrylate)”
BuprenorphineName	Buprenorphine	“The mice were given 0.1 mg/Kg Buprenorphine as an analgesic”
Buprenorphine administrationDose	0.1 mg/Kg	“The mice were given 0.1 mg/Kg Buprenorphine as an analgesic”
ketamineName	ketamine	“Mice were anesthetized with ketamine (200 mg/ml)”
xylacineName	xylacine	“xylacine (50 mg/ml)”
paraformaldehyde (PFA)Name	paraformaldehyde (PFA)	“perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB)”
phosphate buffer (PB)Name	phosphate buffer (PB)	“perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB)”
VT 1000S vibratomeManufacturer	Leica	“VT 1000S, Leica”
blocking solutionName	blocking solution	“incubated for 30 min in blocking solution at room temperature (3% goat serum in TTBS)”
goat serumName	goat serum	“3% goat serum in TTBS”
TTBSName	TTBS	“3% goat serum in TTBS”
PBSName	PBS	“washing three times in PBS (5 min)”
DAPIManufacturer	Life Technology, Carlsbad, USA	“DAPI (2 μM; Life Technology, Carlsbad, USA)”
DAPIName	DAPI	“nuclei were stained with DAPI (2 μM; Life Technology, Carlsbad, USA)”
rabbit anti-Piezo2 antibodyManufacturer	Novus Biological, Littleton, USA	“rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological, Littleton, USA)”
rabbit anti-Piezo2 antibodyName	rabbit anti-Piezo2	“rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological, Littleton, USA)”
rabbit anti-Piezo2Catalog number	NBP1-78624	“rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological, Littleton, USA)”
chicken anti-GFP antibodyManufacturer	Abcam, Cambridge, UK	“chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK)”
chicken anti-GFP antibodyName	chicken anti-GFP	“chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK)”
chicken anti-GFPCatalog number	AB13970	“chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK)”
goat-anti rabbit AlexaFluor-594Name	goat-anti rabbit AlexaFluor-594	“goat-anti rabbit AlexaFluor-594 (1:1000; A11012)”
goat-anti rabbit AlexaFluor-594Catalog number	A11012	“goat-anti rabbit AlexaFluor-594 (1:1000; A11012)”
goat-anti chicken AlexaFluor-488Manufacturer	Life Technology	“goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology)”
goat-anti chicken AlexaFluor-488Name	goat-anti chicken AlexaFluor-488	“goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology)”
goat-anti chicken AlexaFluor-488Catalog number	A11039	“goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology)”
balance beamName	balance beam	“Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top.”
table topName	table top	“beam suspended on two poles 50 cm above a table top”
lampName	lamp	“a lamp above the start point served as an aversive stimulus”
tripodName	tripod	“A video camera on a tripod was used”
video cameraName	video camera	“A video camera on a tripod was used to record the time required to cross the beam.”
metal barName	metal bar	“For two limb hanging we used a 2 mm diameter metal bar mounted between two ring stands 37 cm above a cotton pad”
ring standsName	ring stands	“metal bar mounted between two ring stands”
cotton padName	cotton pad	“cotton pad that was situated under the wire”
rotarodManufacturer	Ugo Basile	“rotarod test (Ugo Basile)”
rotarodName	rotarod	“In the rotarod test (Ugo Basile)”
rotating cylinderName	rotating cylinder	“the mice were placed on a 5 cm diameter rotating cylinder”
CatWalk system 7.1Manufacturer	Noldus Information Technology	“CatWalk system 7.1 (Noldus Information Technology)”
CatWalk system 7.1Name	CatWalk system 7.1	“CatWalk system 7.1 (Noldus Information Technology)”
CatWalk systemVersion	7.1	“CatWalk system 7.1 (Noldus Information Technology)”
glass chamberName	glass chamber	“the mice were placed in a glass chamber (85 cm long and 8.5 cm wide)”
fluorescent lightName	fluorescent light	“Fluorescent light is emitted inside the apparatus”
CatWalk softwareName	CatWalk software	“analyzed with the CatWalk software”
Clampex 10.1Name	Clampex 10.1	“The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA)”
Origin 7.5Name	Origin 7.5	“Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA)”
SigmaStatName	SigmaStat	“Statistical analyses were performed with SigmaStat (Systat Software Inc.)”

Procedure (16)

A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons

Florez-Paz D, Bali KK, Kuner R, et al. Scientific reports 2016;6:25923.

The test records were read three times by AI and checked by code. No person has reviewed them yet. How records are made

Balance beam test

Compare with 4 other papers

Setting	Value	Paper’s words
Beam size	1 m long, 2 cm wide	“Balance was tested on a 1 m long, 2 cm wide beam”
Beam height	50 cm	“beam suspended on two poles 50 cm above a table top”
Number of trials	Unclear at least three times One passage covers all tests at once. It gives a lower bound on repetitions per animal but does not say whether these fell within one session or day or across sessions.	“each of which was repeated at least three times in each animal”
Other settings in the paper
Aversive stimulus	a lamp above the start point	“a lamp above the start point served as an aversive stimulus”
Box at the finish point	Food	“Food was placed in a box at the finish point to attract the mouse”
Apparatus
Balance beam		“Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top. Food was placed in a box at the finish point to attract the mouse and a lamp above the start point served as an aversive stimulus. A video camera on a tripod was used to record the time required to cross the beam.”

Gait analysis

Compare with 8 other papers

Setting	Value	Paper’s words
Walkway size	85 cm long and 8.5 cm wide	“the mice were placed in a glass chamber (85 cm long and 8.5 cm wide) and they were allowed to move freely across the walkway”
Number of runs	at least three times	“scored all the tests, each of which was repeated at least three times in each animal” “the values from the individual tracks were averaged”
Not in the Methods	Run duration criterion
Apparatus
CatWalk system 7.1	Noldus Information Technology	“The animal’s gait was tested with the CatWalk system 7.1 (Noldus Information Technology), where the mice were placed in a glass chamber (85 cm long and 8.5 cm wide) and they were allowed to move freely across the walkway.”
CatWalk software		“allowing the paw prints to be captured by a video camera and analyzed with the CatWalk software”

Rotarod test

Compare with 9 other papers

Setting	Value	Paper’s words
Rod speed	5 rpm and it accelerated to 20 rpm	“The initial speed of rotation was 5 rpm and it accelerated to 20 rpm”
Number of trials	Unclear at least three times One passage gives one repetition count for all tests, including the rotarod, as repetitions per animal. It does not say whether these were trials within one session or day or repeats across sessions, and it is only a lower bound.	“all the tests, each of which was repeated at least three times in each animal”
Not in the Methods	Trial duration
Other settings in the paper
Diameter rotating cylinder	5 cm	“the mice were placed on a 5 cm diameter rotating cylinder”
Apparatus
Rotarod	Ugo Basile	“In the rotarod test (Ugo Basile), the mice were placed on a 5 cm diameter rotating cylinder along which they must walk in order not to fall off. The initial speed of rotation was 5 rpm and it accelerated to 20 rpm.”

Facts checked by a reviewer

A person went through this paper’s whole Methods section, beyond the behavioral tests, and confirmed these 118 facts against the text. No person has checked the test records above.

Study design (1)

Item	Value	Paper’s words
This studyBlinding	The same investigator scored all the tests	“The same investigator scored all the tests”

Subjects and groups (13)

Item	Value	Paper’s words
Experiment subjectsSpecies	mouse	“Adult C57Bl/6J mice were used in this study.”
Experiment subjectsStrain	C57Bl/6J	“Adult C57Bl/6J mice were used in this study.”
Experiment subjectsAge	6–9 week-old	“6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice”
Experiment subjectsSex	male	“Male animals were used for in vitro recordings”
Experimental miceAge	6–9 week-old	“we tested a cohort of 6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice”
Pvalb-Cre:RCE:Piezo2cKO miceLabel	Pvalb-Cre:RCE:Piezo2cKO mice	“we tested a cohort of 6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice (n = 7)”
Pvalb-Cre:RCE:Piezo2cKO miceN allocated	7	“Pvalb-Cre:RCE:Piezo2cKO mice (n = 7)”
Behavioral test subjectsAge	6–9 week-old	“a cohort of 6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice (n = 7) and their control littermates (n = 7)”
Behavioral test subjectsSex	female	“6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice”
Pvalb-Cre:RCE:Piezo2cKO groupLabel	Pvalb-Cre:RCE:Piezo2cKO	“6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice (n = 7)”
Pvalb-Cre:RCE:Piezo2cKO groupN allocated	7	“6–9 week-old female Pvalb-Cre:RCE:Piezo2cKO mice (n = 7)”
control littermates groupLabel	control littermates	“and their control littermates (n = 7)”
control littermates groupN allocated	7	“and their control littermates (n = 7)”

Materials and equipment (88)

Item	Value	Paper’s words
vibrating slicerName	vibrating slicer	“the brainstem was attached at its rostral end to the platform of the vibrating slicer”
vibrating slicerModel	series 1000	“the vibrating slicer (Pelco, TPI, series 1000, St. Louis, USA)”
isofluraneName	isoflurane	“animals were anesthetized by isoflurane inhalation”
DiI tracerManufacturer	Life Technology, Carlbad, USA	“the DiI tracer (5%: Life Technology, Carlbad, USA)”
DiI tracerName	DiI tracer	“the DiI tracer (5%: Life Technology, Carlbad, USA) was injected”
cyanoacrylateName	cyanoacrylate	“The incision was closed with a drop of cyanoacrylate”
artificial cerebrospinal fluid (ACSF)Name	artificial cerebrospinal fluid (ACSF)	“immersed in ice-cold, oxygenated (95% O2–5% CO2) artificial cerebrospinal fluid (ACSF)”
ACSFName	artificial cerebrospinal fluid (ACSF)	“artificial cerebrospinal fluid (ACSF)”
Multiclamp 700 amplifierName	Multiclamp 700 amplifier	“using a Multiclamp 700 amplifier”
pCLAMP 10Name	pCLAMP 10	“data were acquired using pCLAMP 10 software”
pCLAMP 10Version	10	“the data were acquired using pCLAMP 10 software”
Digidata 1440Manufacturer	Molecular Devices, Sunnyvale, USA	“Digidata 1440 (Molecular Devices, Sunnyvale, USA)”
Digidata 1440Name	Digidata 1440	“and a Digidata 1440 (Molecular Devices, Sunnyvale, USA)”
borosilicate glass capillariesName	borosilicate glass capillaries	“Patch electrodes of 4–7 MΩ were pulled from borosilicate glass capillaries”
patch electrodesName	patch electrodes	“Patch electrodes of 4–7 MΩ were pulled from borosilicate glass capillaries”
internal solutionName	internal solution	“The pipettes were filled with an internal solution containing (in mM): 115 K-gluconate, 25 KCl, 9 NaCl, 10 HEPES, 0.2 EGTA, 1 MgCl2, 3 K2-ATP and 1 Na-GTP, pH 7.2 adjusted with KOH and 280–290 mosmol Kg−1.”
external control solutionName	external control solution	“The control external solution consisted of (in mM): 140 NaCl, 3 KCl, 1 CaCl2 2 MgCl2, 10 glucose and 10 HEPES (pH 7.2).”
recording chamberName	recording chamber	“Slices were transferred to a recording chamber and perfused with oxygenated external control solution.”
fluorescence microscopeName	fluorescence microscopy (Olympus BX50WI)	“fluorescence microscopy (Olympus BX50WI)”
fluorescence microscopeManufacturer	Olympus	“fluorescence microscopy (Olympus BX50WI)”
Imago cameraManufacturer	Till photonics GmbH, Germany	“an Imago camera (both Till photonics GmbH, Germany)”
Imago cameraName	Imago camera	“an Imago camera (both Till photonics GmbH, Germany)”
Imaging Workbench 6.0Name	Imaging Workbench 6.0	“the Imaging Workbench 6.0 software (INDEC BioSystems)”
Imaging Workbench 6.0Manufacturer	INDEC BioSystems	“the Imaging Workbench 6.0 software (INDEC BioSystems)”
Imaging Workbench 6.0Version	6.0	“the Imaging Workbench 6.0 software (INDEC BioSystems)”
heat-closed glass pipetteName	heat-closed glass pipette	“using a heat-closed glass pipette driven by a MM3A-LS piezoelectric micromanipulator”
MM3A-LS piezoelectric micromanipulatorManufacturer	Kleindiek, Reutlingen, Germany	“MM3A-LS piezoelectric micromanipulator (Kleindiek, Reutlingen, Germany)”
MM3A-LS piezoelectric micromanipulatorName	MM3A-LS piezoelectric micromanipulator	“MM3A-LS piezoelectric micromanipulator (Kleindiek, Reutlingen, Germany)”
Piezo2 shRNACatalog number	TR509519	“Cat. No. TR509519: Origene, USA”
Piezo2 shRNAManufacturer	Origene, USA	“Cat. No. TR509519: Origene, USA”
AAV backbone PAM vectorName	AAV backbone of the PAM vector	“adeno-associated viral (AAV) backbone of the PAM vector”
U6 promoterName	U6 promoter	“along with the U6 promoter”
GFP reporter cassetteName	GFP reporter cassette	“The expression of the native GFP reporter cassette in the final clones was confirmed by the strong GFP signal following transfection into HEK-293 cells”
HEK-293 cellsName	HEK-293 cells	“following transfection into HEK-293 cells”
rAAV serotype 2/8 particlesName	rAAV serotype 2/8 particles	“The rAAV serotype 2/8 particles carrying shRNA against Piezo2 (AAV-Piezo2-sh-1) or a scrambled shRNA were generated in house by co-transfecting HEK-293 cells with the cloned rAAV backbone plasmid and the helper 2/8 plasmids”
AAV-Piezo2-sh-1Name	AAV-Piezo2-sh-1	“rAAV serotype 2/8 particles carrying shRNA against Piezo2 (AAV-Piezo2-sh-1)”
scrambled shRNA virusName	scrambled shRNA	“or a scrambled shRNA were generated”
Nanoliter2010Manufacturer	WPI	“nanoliter injector microprocessor (Nanoliter2010, WPI)”
glass micropipetteName	glass micropipette	“The virus was delivered using a glass micropipette”
nanoliter injector microprocessorName	Nanoliter2010	“nanoliter injector microprocessor (Nanoliter2010, WPI)”
Nanoliter2010 injectorManufacturer	WPI	“nanoliter injector microprocessor (Nanoliter2010, WPI)”
AAV-Piezo2-sh-1 injectionVolume	700 nl	“the viral solution was injected in a total volume of 700 nl”
bonewaxName	bonewax	“a layer of adhesive cement (bonewax) was applied around the hole”
cyanocrylateName	cyanocrylate	“We then sutured the skin with a tissue adhesive (cyanocrylate)”
BuprenorphineName	Buprenorphine	“The mice were given 0.1 mg/Kg Buprenorphine as an analgesic”
Buprenorphine administrationDose	0.1 mg/Kg	“The mice were given 0.1 mg/Kg Buprenorphine as an analgesic”
ketamineName	ketamine	“Mice were anesthetized with ketamine (200 mg/ml)”
xylacineName	xylacine	“xylacine (50 mg/ml)”
paraformaldehyde (PFA)Name	paraformaldehyde (PFA)	“perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB)”
phosphate buffer (PB)Name	phosphate buffer (PB)	“perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB)”
VT 1000S vibratomeManufacturer	Leica	“VT 1000S, Leica”
blocking solutionName	blocking solution	“incubated for 30 min in blocking solution at room temperature (3% goat serum in TTBS)”
goat serumName	goat serum	“3% goat serum in TTBS”
TTBSName	TTBS	“3% goat serum in TTBS”
PBSName	PBS	“washing three times in PBS (5 min)”
DAPIManufacturer	Life Technology, Carlsbad, USA	“DAPI (2 μM; Life Technology, Carlsbad, USA)”
DAPIName	DAPI	“nuclei were stained with DAPI (2 μM; Life Technology, Carlsbad, USA)”
rabbit anti-Piezo2 antibodyManufacturer	Novus Biological, Littleton, USA	“rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological, Littleton, USA)”
rabbit anti-Piezo2 antibodyName	rabbit anti-Piezo2	“rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological, Littleton, USA)”
rabbit anti-Piezo2Catalog number	NBP1-78624	“rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological, Littleton, USA)”
chicken anti-GFP antibodyManufacturer	Abcam, Cambridge, UK	“chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK)”
chicken anti-GFP antibodyName	chicken anti-GFP	“chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK)”
chicken anti-GFPCatalog number	AB13970	“chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK)”
goat-anti rabbit AlexaFluor-594Name	goat-anti rabbit AlexaFluor-594	“goat-anti rabbit AlexaFluor-594 (1:1000; A11012)”
goat-anti rabbit AlexaFluor-594Catalog number	A11012	“goat-anti rabbit AlexaFluor-594 (1:1000; A11012)”
goat-anti chicken AlexaFluor-488Manufacturer	Life Technology	“goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology)”
goat-anti chicken AlexaFluor-488Name	goat-anti chicken AlexaFluor-488	“goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology)”
goat-anti chicken AlexaFluor-488Catalog number	A11039	“goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology)”
balance beamName	balance beam	“Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top.”
table topName	table top	“beam suspended on two poles 50 cm above a table top”
lampName	lamp	“a lamp above the start point served as an aversive stimulus”
tripodName	tripod	“A video camera on a tripod was used”
video cameraName	video camera	“A video camera on a tripod was used to record the time required to cross the beam.”
metal barName	metal bar	“For two limb hanging we used a 2 mm diameter metal bar mounted between two ring stands 37 cm above a cotton pad”
ring standsName	ring stands	“metal bar mounted between two ring stands”
cotton padName	cotton pad	“cotton pad that was situated under the wire”
rotarodManufacturer	Ugo Basile	“rotarod test (Ugo Basile)”
rotarodName	rotarod	“In the rotarod test (Ugo Basile)”
rotating cylinderName	rotating cylinder	“the mice were placed on a 5 cm diameter rotating cylinder”
CatWalk system 7.1Manufacturer	Noldus Information Technology	“CatWalk system 7.1 (Noldus Information Technology)”
CatWalk system 7.1Name	CatWalk system 7.1	“CatWalk system 7.1 (Noldus Information Technology)”
CatWalk systemVersion	7.1	“CatWalk system 7.1 (Noldus Information Technology)”
glass chamberName	glass chamber	“the mice were placed in a glass chamber (85 cm long and 8.5 cm wide)”
fluorescent lightName	fluorescent light	“Fluorescent light is emitted inside the apparatus”
CatWalk softwareName	CatWalk software	“analyzed with the CatWalk software”
Clampex 10.1Name	Clampex 10.1	“The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA)”
Origin 7.5Name	Origin 7.5	“Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA)”
SigmaStatName	SigmaStat	“Statistical analyses were performed with SigmaStat (Systat Software Inc.)”

Procedure (16)

Item	Value	Paper’s words
Anesthetize animals for dye injectionAction	animals were anesthetized by isoflurane inhalation	“animals were anesthetized by isoflurane inhalation”
Allow DiI diffusionAction	the DiI was allowed to diffuse retrogradely into the cell bodies in the MTN	“the DiI was allowed to diffuse retrogradely into the cell bodies in the MTN”
Attach brainstem to vibrating slicerAction	The brainstem was attached at its rostral end to the platform of the vibrating slicer (Pelco, TPI, series 1000, St. Louis, USA) and covered with ice-cold ACSF	“The brainstem was attached at its rostral end to the platform of the vibrating slicer (Pelco, TPI, series 1000, St. Louis, USA) and covered with ice-cold ACSF”
Apply mechanical stepsAction	A series of mechanical steps in 1 μm increments were applied every 10 s	“A series of mechanical steps in 1 μm increments were applied every 10 s”
Apply adhesive cement and sutureAction	a layer of adhesive cement (bonewax) was applied around the hole. We then sutured the skin with a tissue adhesive (cyanocrylate)	“a layer of adhesive cement (bonewax) was applied around the hole. We then sutured the skin with a tissue adhesive (cyanocrylate)”
Administer Buprenorphine and recoveryAction	The mice were given 0.1 mg/Kg Buprenorphine as an analgesic and they remained on a heated pad until they recovered from the anesthesia	“The mice were given 0.1 mg/Kg Buprenorphine as an analgesic and they remained on a heated pad until they recovered from the anesthesia”
Anesthetize for perfusionAction	Mice were anesthetized with ketamine (200 mg/ml) and xylacine (50 mg/ml)	“Mice were anesthetized with ketamine (200 mg/ml) and xylacine (50 mg/ml)”
Apply secondary antibodiesAction	the secondary antibodies were applied for 2 h at room temperature	“the secondary antibodies were applied for 2 h at room temperature”
Animal housingParameter	12 h light/dark cycle at 21 °C with food and water available ad libitum	“The animals were housed on a 12 h light/dark cycle at 21 °C with food and water available ad libitum.”
Balance beam testAction	Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top. Food was placed in a box at the finish point to attract the mouse and a lamp above the start point served as an aversive stimulus. A video camera on a tripod was used to record the time required to cross the beam.	“Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top. Food was placed in a box at the finish point to attract the mouse and a lamp above the start point served as an aversive stimulus. A video camera on a tripod was used to record the time required to cross the beam.”
Behavioral test: balance beamAction	Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top	“Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top”
Behavioral test: balance beamParameter	1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top	“Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top”
Behavioral test: CatWalk gait analysisAction	The animal’s gait was tested with the CatWalk system 7.1 (Noldus Information Technology)	“The animal’s gait was tested with the CatWalk system 7.1 (Noldus Information Technology)”
Analyze regularity indexAction	We analyzed the regularity index (%) that expresses the number of normal step sequence patterns relative to the total number of paw placements, a fractional measure of inter-paw coordination	“We analyzed the regularity index (%) that expresses the number of normal step sequence patterns relative to the total number of paw placements, a fractional measure of inter-paw coordination”
Behavioral test scoringAction	The same investigator scored all the tests, each of which was repeated at least three times in each animal, and the values from the individual tracks were averaged.	“The same investigator scored all the tests, each of which was repeated at least three times in each animal, and the values from the individual tracks were averaged.”
Animal habituation and trainingAction	All the animals were habituated and trained in the different tests prior to performing the experiment.	“All the animals were habituated and trained in the different tests prior to performing the experiment.”