Journal: Journal of Cell Science

Article Title: Piezo1-induced durotaxis of pancreatic stellate cells depends on TRPC1 and TRPV4 channels

doi: 10.1242/jcs.263846

Figure Lengend Snippet: Multiple mechanosensitive channels are necessary for durotaxis. (A) Durotaxis polar plots depict cell trajectories over 24 h (black lines) of wild-type (Wt, top) and TRPC1 KO (TRPC1-KO, bottom) PSCs treated with the TRPV4 inhibitor 100 nM HC067047. For each plot, the gradient orientation is towards the left side. The radii of the blue half circles on the right-hand and left-hand sides of each plot are proportional to the mean cellular displacement towards 0° and 180°, respectively. Radial lines indicate 0°, 90°, 180° and 270°. Scale bar: 100 µm for the migration trajectories and 50 µm for the half circles. Radius of the concentric gray circle is a visual aid for the scale bar. n =28 for Wt and n =59 for TRPC1-KO cell trajectories from N =3 experiments. (B) Durotaxis indices of n =28 wild-type PSCs (TRPC1 +) from N =3 experiments and n =59 PSCs from TRPC1 KO mice (TRPC1 −) from N =3 experiments (left and right from vertical line, respectively) seeded on gradient hydrogels treated with 0.1% DMSO (black), 10 µM TRPV4 activator GSK1016790A (TRPV4 ↑, light blue) or 100 nM HC067047 (TRPV4 ↓, purple). They are compared with wild-type PSCs seeded on gels with homogeneous stiffness (gray, median marked by dotted line). n =50 cell trajectories from N =4 experiments. (C) Representative graphs of Mn 2+ quench experiments showing the relative fluorescence intensity (F/F 0 ) of Fura-2 AM-loaded wild-type and TRPC1 KO PSCs. Upon application of Mn 2+ (box), quenching of the Fura-2 signal can be observed under control conditions (black), in the presence of 20 nM GSK1016790A (blue) or 2 µM HC067047 (purple). (D) Mn 2+ entry rates of wild-type PSCs (TRPC1 +) and PSCs from TRPC1 KO mice (TRPC1 −) determined under control conditions (TRPV4 +) and in the presence of GSK1016790A (20 nM, TRPV4 ↑; 100 nM, TRPV4 ↑↑) or 2 µM HC067047 (TRPV4 ↓), as indicated. n ≥87 cells from N =3 experiments. (E) Representative immunofluorescence image of a PSC stained for TRPV4 (red) and TLN1 (cyan), with magnified image in the inset. Scale bars: 50 µm; inset, 5 µm. Images are representative of N =3 experiments. (F) Durotaxis indices as a function of ion channel activity. To estimate total channel activity, individual channel activity was binned: impaired channel=0; intermediate activity=1 for TRPV4 and TRPC1; intermediate activity=2 for Piezo1; overactivation=3 for TRPV4; and overactivation=4 for Piezo1. A Gaussian curve was fitted over the data. Data in B are represented as median±95% confidence interval; data in D are represented as mean±s.e.m.; data in F are represented as median±95% confidence interval. Statistical tests: Kruskal–Wallis statistical test with Dunn's post-hoc test in B; one-way ANOVA followed by Tukey's post-hoc test in D. Statistical comparison of panel B is further detailed in Table S2 . a.u., arbitrary units.

Article Snippet: For TRPV4 and Talin-1 immunostaining, PSCs were fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 at 4°C for 20 min. After washing twice with PBS, samples were blocked using 10% FCS (Sigma-Aldrich) in PBS at 4°C for 1 h. Subsequently, primary antibodies against αSMA (1:200; A2547, RRID: AB_476701; Sigma-Aldrich, Merck KGaA), vimentin (1:500; 10366-1-AP, RRID: AB_2273020; Proteintech), TRPV4 (1:200; ACC-034, RRID: AB_2040264; Alomone, RRID: AB_2534069) and Talin-1 (1:500; T3287, RRID:AB_477572; Sigma-Aldrich) were added to the gels at 4°C for 2 h. After washing three times with PBS, Alexa Fluor 488-conjugated secondary antibodies against mouse IgG (1:500; RRID: AB_2534069, Invitrogen) and Cy3-conjugated antibodies against rabbit IgG (1:500; RRID: AB_10563288, Invitrogen), as well as DAPI (1:100,000, Sigma-Aldrich), were added at 4°C for 20 min. An Axio Observer Z.1 inverted microscope (Carl Zeiss AG) equipped with a CMOS camera and 40× objective (400× total magnification, Carl Zeiss AG) objective was used to acquire the immunofluorescence images.

Techniques: Migration, Fluorescence, Control, Immunofluorescence, Staining, Activity Assay, Comparison

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet: Flow cytometry enriched antibody cocktail (22 μL) panel for analyzing rarer granulocyte and myeloid cells in mouse spleen for downstream scRNA-seq

Article Snippet: FcεRIα Antibody, anti-mouse, PE-Vio® 615, REAfinityTM Clone REA1079 , Miltenyi Biotec , Cat# 130-118-899; RRID: AB_2801723.

Techniques: Flow Cytometry, Marker

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet:

Article Snippet: FcεRIα Antibody, anti-mouse, PE-Vio® 615, REAfinityTM Clone REA1079 , Miltenyi Biotec , Cat# 130-118-899; RRID: AB_2801723.

Techniques: Recombinant, Sterility, Saline, Extraction, Blocking Assay, Software, Solvent, Sequencing, RNA Sequencing, Transferring, Aerosol, Irradiation, Inverted Microscopy, Flow Cytometry, Fluorescence