Journal: bioRxiv

Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

doi: 10.1101/2021.01.20.427483

Figure Lengend Snippet: Several bands around 310-150KDa are detected by immunoblot (IB) in the lysates of neuronal cell lines, which are eliminated by preincubation with blocking peptide (BP) ( A ). In DRG lysate, the antibody detects several bands at approximately 310, 240, 180, and 130 KDa and marked as putative “isoforms (iso)” 1-4, respectively, of the target protein since they are eliminated by preincubation with BP ( B ). Piezo2 IB in the cytosolic and membrane fractions prepared from pooled L4/L5 DRG show that the putative Piezo2 iso2 (∼240KDa) and iso3 (∼180KDa) are more enriched in membranes while the canonical Piezo2 (KDa ∼310) and putative iso4 (KDa ∼130) are more localized in cytosol ( C ). Representative images of double immunostaining (IS) of Piezo2 (red) and Tubb3 (green) on DRG sections reveal pan-neuronal Piezo2-IR ( D ) and preincubation with BP eliminated the staining ( D1 ). Representative images of double-IS of Piezo2 (red) and Tubb3 (green) on trigeminal ganglia (TG) sections reveal pan-neuronal Piezo2-IR ( E ). Scale bars: 100 μm for all. Expression plasmid map ( F ) in which U6 and H1 promoters transcribe convergent apposing Piezo2-shRNA expression. The Piezo2-shRNA cassette (sequences shown on the top of plasmid map) is cloned into Mlul site (pointed by arrowhead) of plasmid and named pU6/H1-Piezo2shRNA-CMV-EGFP (Piezo2-shRNA). Transfection of Piezo2-shRNA into N2A cells yielded 70-80% transfection rate (n=4) ( G ); Piezo2 protein in N2A lysates analyzed by IB using ProSci Piezo2 antibody: Lane 1, mock transfection; lane 2, transfection with scrambled RNA; lane 3-4, duplicate transfection using Piezo2-shRNA ( H ); Densitometry of IBs of fold change in Piezo2 protein (left) and qPCR (right) show fold change in Piezo2 mRNA (**p<0.01, one-way ANOVA, Turkey post-hoc comparison) ( I ). Representative traces ( J ) and mean current density in response to mechanical force ramp ( K ) shows that Piezo2 knockdown significantly decreased mechanically-evoked rapid-adapt inward currents, compared to controls. ***p<0001; two-way ANOVA of main effects of groups with Bonferroni post-hoc comparison. #, ## , and ### denote p<0.05, <0.01, and<0.001, respectively, between groups.

Article Snippet: Selectivity of the ProSci Piezo2 antibody was further validated by comparing the relevant immunoblot Piezo2 protein level in the control N2A cells versus the N2A cells in which the RNAi induced Piezo2 knockdown, as was verified by qPCR and patch-recording of Piezo2 channel activity.

Techniques: Western Blot, Blocking Assay, Membrane, Double Immunostaining, Staining, Expressing, Plasmid Preparation, shRNA, Clone Assay, Transfection, Comparison, Knockdown

Journal: bioRxiv

Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

doi: 10.1101/2021.01.20.427483

Figure Lengend Snippet: Representative montage images of DRG sections show Piezo2-IR (red) co-stained with a selection of DRG neuronal markers (green), including Tubb3 ( A ), IB4 ( B ), CGRP ( C ), NF200 ( D ), TrkB ( E ), and NKA1 α ( F ) with the squared region shown at high magnification ( F1 ). The panels in the right-side of A - E calculate the percentage of Piezo2-neurons overlaid to Tubb3-neurons ( A 1), as well as neurons positive for IB4 ( B1 ), CGRP ( C1 ), NF200 ( D1 ), and TrkB ( E1 ) overlaid to Piezo2-neurons. The numbers are the counted Piezo2-IR neurons (red) and marker-labeled neurons (green). ICA analyzes colocalization between Piezo2 and NKA1 α by an ImageJ 1.46r software plugin colocalization analysis module ( G ). Scatter plots for the region demarcated by the white dashed line in F1 panel show data clustered along both positive and negative axes for both Piezo2 and NKA1 α . “A” is the intensity of Piezo2 while “a” is the average of these values, and “B” is the intensity of NKA1a while “b” is the average of these values. For this region, the ICQ value is 0.127 (P sign test <0.001), indicating partial immunocolocalization. Representative montage images show Piezo2 (red) with a selection of glial cell markers (green), including S100 ( H, I ), MBP ( J ), GS ( K ) with the squared region shown at high magnification ( K1 ), and GFAP ( L ) with the squared region shown at high magnification ( L1 ). White arrowheads in panel I, K1 , and L1 point to the immune-colabeled glial cells. ICA analysis for colocalization between Piezo2 and GFAP for the region demarcated by the white dashed line in L1 panel show scattered plot data clustered along both positive and negative axes for both Piezo2 and GFAP. “A” is the intensity of Piezo2 while “a” is the average of these values, and “B” is the intensity of GFAP while “b” is the average of these values. For this region, the intensity correlation quotient (ICQ) value is 0.205 (P sign test <0.001), indicating partial immunocolocalization ( M ). Scale bars: 50 μm for all.

Article Snippet: Selectivity of the ProSci Piezo2 antibody was further validated by comparing the relevant immunoblot Piezo2 protein level in the control N2A cells versus the N2A cells in which the RNAi induced Piezo2 knockdown, as was verified by qPCR and patch-recording of Piezo2 channel activity.

Techniques: Staining, Selection, Marker, Labeling, Software

Journal: bioRxiv

Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

doi: 10.1101/2021.01.20.427483

Figure Lengend Snippet: Representative montage images on sciatic nerve cryosections show double immunostaining (IS) of Piezo2 (red) with a selection of neuronal markers (green), including Tubb3 ( A ), NF200 ( B ), IB4 ( C ), and CGRP ( D ). Representative montage images on sciatic nerve cryosections show double-IS of Piezo2 (red) with glia markers (green), including S100 ( E ), p75NTR ( F ), GFAP ( G ), and MBP ( H ). Representative montage images on human Schwann cells ( I ) and isolated Schwann cells ( J ) from rat sciatic nerve show double-IS of Piezo2 (red) with S100 (green). Scale bars: 50 μm for all.

Article Snippet: Selectivity of the ProSci Piezo2 antibody was further validated by comparing the relevant immunoblot Piezo2 protein level in the control N2A cells versus the N2A cells in which the RNAi induced Piezo2 knockdown, as was verified by qPCR and patch-recording of Piezo2 channel activity.

Techniques: Double Immunostaining, Selection, Isolation

Journal: bioRxiv

Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

doi: 10.1101/2021.01.20.427483

Figure Lengend Snippet: Representative IHC image shows detection of Piezo2-IR (red) in the spinal cord neurons at all laminae ( A ), with grey matter pseudocolored in blue and a inset showing magnified image, counterstained with Hoechst (blue), of spinal ventral horn ( A1 ). Representative montage images of double-IS on the DH regions reveal Piezo2-IR (red) with NeuN (green) ( B ) and a selection of dorsal horn presynaptic markers (green), including IB4 ( C ), CGRP ( D ), Syp ( E ), and Synpr ( F ), showing immunocolocalization (yellow) in the marge images. Insets in the merged images of panels C and D are higher magnifications of colabeling of Piezo2 (red) with IB4 or CGRP (green) with nuclear counterstained by Hoechst (blue); arrowheads pointing to colocalization (yellow). Representative montage images show negative Piezo2 in GFAP-positive astrocytes ( G ) and Iba1-positive microglia ( H ) of spinal cord sections. The regions within the squares in panels E - H are shown at high magnification ( E1 - H1 ). Scale bars: 50 μm for all.

Article Snippet: Selectivity of the ProSci Piezo2 antibody was further validated by comparing the relevant immunoblot Piezo2 protein level in the control N2A cells versus the N2A cells in which the RNAi induced Piezo2 knockdown, as was verified by qPCR and patch-recording of Piezo2 channel activity.

Techniques: Selection

Journal: bioRxiv

Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

doi: 10.1101/2021.01.20.427483

Figure Lengend Snippet: Representative montage images of hindpaw glabrous skin sections display double immunostaining (double-IS) of Piezo2 (red) with CK14 (green), a maker of epidermal basal layer keratinocytes and Merkel cells, showing colabeling (yellow, empty arrowheads) in the merged image ( A ) with the squared regions shown at high magnification ( A1 ). Representative montage images reveal Piezo2-IR in Meissner’s corpuscles (white arrowhead) and immunocolocalization with CK14-positive Merkel cells (empty arrowheads) ( B ). Representative montage images show immunocolocalization of Piezo2-IR with Tubb3 ( C ), NF200 ( D ), IB4 ( E ), CGRP ( F ), TrkB ( G ), and S100 ( H ) in the nerve bundles within dermis. Representative montage images show immunocolocalization of Piezo2-IR with S100 in epidermal basal layer cells ( I , empty arrowheads. White arrowheads point to Meissner’s corpuscles), with the squared regions shown at high magnification ( I1 ). Representative montage images show immunocolocalization of Piezo2-IR with S100 in Meissner’s corpuscles (white arrowheads) ( J ). Human primary epidermal melanocytes ( K ) show Piezo2-IR ( L ) which is completely eliminated by blocking peptide preabsorption before ICC ( M ) and colabeled with S100 ( N ). Scale bars: 25 μm for all.

Article Snippet: Selectivity of the ProSci Piezo2 antibody was further validated by comparing the relevant immunoblot Piezo2 protein level in the control N2A cells versus the N2A cells in which the RNAi induced Piezo2 knockdown, as was verified by qPCR and patch-recording of Piezo2 channel activity.

Techniques: Double Immunostaining, Blocking Assay

Journal: bioRxiv

Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

doi: 10.1101/2021.01.20.427483

Figure Lengend Snippet: RT-PCR shows amplification of Piezo1 and Piezo2 transcripts in rat 50B11 cells ( A ), rat DRG ( B ), rat primary cultured SGCs at day of in vitro 4 (DIV, C ), and rat primary cultured Schwann cells at DIV5 ( D ) by two different primer pairs specific for amplification of rat Piezo1 and Piezo2 transcripts, as well as detection of Piezo1 and Piezo2 transcripts by two different primer pairs specific for human Piezo1 and 2 in human primary cultured melanocytes ( E ). Lane 1: ladder, lane 2-3: Piezo1 amplified by two piezo1 primer pairs ( A-D , rat; E , human), lane 3-4: Piezo2 amplified by two piezo2 primer pairs ( A-D , rat; E , human), lane 7: Gapdh, and lane 8: negative control.

Article Snippet: Selectivity of the ProSci Piezo2 antibody was further validated by comparing the relevant immunoblot Piezo2 protein level in the control N2A cells versus the N2A cells in which the RNAi induced Piezo2 knockdown, as was verified by qPCR and patch-recording of Piezo2 channel activity.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Cell Culture, In Vitro, Negative Control

Journal: bioRxiv

Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

doi: 10.1101/2021.01.20.427483

Figure Lengend Snippet: CFA rats developed mechanical allodynia (vF) and hyperalgesia (Pin) A ); *** denotes p <0.001, unpaired, two-tailed Student’s t -test for vF and Mann–Whitney test for Pin, compared between groups after CFA. NKA1 α -deficient cytosolic fractions and NKA1 α -enriched membrane fractions were extracted from the DRG (pooled L4/L5) at 10 days after CFA or saline injection, and subjected to immunoblotting (IB) as shown in the representative IBs of Piezo2, Iba1, NKA1 α , and Gapdh of cytosol ( B , left) and membrane fractions ( B , right), respectively. The densitometry of canonical piezo2 (∼310KDa, iso1) and putative iso2 (∼250KDa) outlined by a rectangle were analyzed and summarized in bar charts ( C ); *** p <0.001, unpaired, two-tailed Student’s t -test. Piezo2 intensities in DH of saline ( D ) and CFA ( E ) were inverted; the upper and lower threshold optical intensity of Piezo2-IR adjusted to encompass and match the IR that appears in red ( D1 and E1 ) with the rectangles positioned over laminae territory throughout the mediolateral axis on the contralateral (contra.) and ipsilateral (ipsi.) sides, and quantified as described in Method. Scale bars: 100 μm for all. The integrated density (product of area and density) calculated by use of ImageJ, and fold change (ratio of ipsi./contra.) summarized in the bar charts ( F ). *** denotes p <0.001 by two-tailed unpaired Student’s t -test.

Article Snippet: Selectivity of the ProSci Piezo2 antibody was further validated by comparing the relevant immunoblot Piezo2 protein level in the control N2A cells versus the N2A cells in which the RNAi induced Piezo2 knockdown, as was verified by qPCR and patch-recording of Piezo2 channel activity.

Techniques: Two Tailed Test, MANN-WHITNEY, Membrane, Saline, Injection, Western Blot

Journal: bioRxiv

Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

doi: 10.1101/2021.01.20.427483

Figure Lengend Snippet: Bar charts summarize mechanical allodynia (vF) and hyperalgesia (Pin) that encompass various pain models in our previously published studies, as indicated ( A - D ). Representative IHC montage images on the DH show double immunostaining (IS) of Piezo2 (red) with IB4 (green) in control ( E ), SNL ( F ), SNI ( G ), and TNI ( H ) rats, and DH Piezo2-IR intensities from the presentative IHC images are inverted, and optical threshold adjusted encompass and match the IR that appears in red ( E1 - H1 ), respectively; and quantified as described in Method. The rectangles positioned over laminae territory throughout the mediolateral axis on the contralateral (contra.) and ipsilateral (ipsi.) DHs. The grey matters in the merged images are pseudocolored in blue. Representative IHC images of Piezo2 (red) ( I ) and Iba1 ( I1 ) from two adjacent sections from MIA-OA rat display apparent increased Piezo2-IR in parallel with microgliosis at the ipsilateral side to MIA; white dash-lines outline the dorsal horns (left panels), and grey matters pseudocolored in blue (right panels). Scale bars: 200μm for all. Quantitative comparison of DH Piezo2-IR intensity between ipsi. and contra. sides are analyzed by the Image J (see Methods and suppl. Fig. 5), and the integrated intensity (product of area and density) calculated, and fold change (ratio of ipsi./contra.) presented as the bar charts in SNL ( J ), SNI ( K ), TNI ( L ), and MIA ( M ). *** denotes p<0.001 by two-tailed unpaired Student’s t -test.

Article Snippet: Selectivity of the ProSci Piezo2 antibody was further validated by comparing the relevant immunoblot Piezo2 protein level in the control N2A cells versus the N2A cells in which the RNAi induced Piezo2 knockdown, as was verified by qPCR and patch-recording of Piezo2 channel activity.

Techniques: Double Immunostaining, Control, Comparison, Two Tailed Test