Journal: Cells

Article Title: Substrate Stiffness Modulates Renal Progenitor Cell Properties via a ROCK-Mediated Mechanotransduction Mechanism

doi: 10.3390/cells8121561

Figure Lengend Snippet: ROCK activity is required to mediate the effects of stiffness on RPC proliferation, migration and differentiation. ( a , b ) Confocal images of F-actin immunodetection by phalloidin (red), paxillin (green) and nuclei with DAPI counterstain (white) in RPCs cultured on ( a ) 12 kPa and ( b ) 50 kPa substrate treated with vehicle (DMSO, control) and with the Rho kinase inhibitor Y27632. Bars = 25 μm ( c ) Average number of paxillin patches in RPCs cultured on 12 and 50 kPa substrates in control condition and treated with Y27632. At least 20 cells for each condition were analyzed. Box-and-whisker plots: Line = median, box = 25–75%, whiskers = 10–90%. ( d ) Percentage of growth inhibition induced by 10 μM Y27632 treatment of RPCs assessed by MTT assay. Data are mean ± SE (replicate n = 8). ( e ) MSD of RPCs cultured on 50 kPa stiffness in presence or absence of Y27632 (n = 46 and 44 trajectories for 50 kPa and 50 kPa + Y27632, respectively). ( f ) Percentage of inhibition of nephrin expression induced by 10 μM Y27632 treatment. ( g ) Percentage of inhibition of Klf15 expression induced by 10 μM Y27632 treatment. Data are expressed as mean ± SE. * p < 0.05 using one-way ANOVA followed by Tukey’s or Bonferroni post-hoc test, as appropriate.

Article Snippet: The following antibodies were used: Anti-paxillin (ab32084, dilution 1:250, Abcam, Cambridge, UK), anti-nephrin (NMP1-30130Y17-R, dilution 1:200, Novus Biologicals Centennial, CO, USA), anti-ROCK1 (Cat# 202694T02, dilution 1:200, Sino Biological, Wayne, PA, USA), anti-ROCK2 (Cat# HPA044109, dilution 1:30, Sigma Aldrich, Milan, Italy).

Techniques: Activity Assay, Migration, Immunodetection, Cell Culture, Whisker Assay, Inhibition, MTT Assay, Expressing

Journal: Cells

Article Title: Substrate Stiffness Modulates Renal Progenitor Cell Properties via a ROCK-Mediated Mechanotransduction Mechanism

doi: 10.3390/cells8121561

Figure Lengend Snippet: ROCK 1 and ROCK2 mediate the effects of stiffness on RPC proliferation and migration. ( a ) ROCK1 and ROCK2 mRNA expression in scramble and LNA-GapmeRs treated RPCs, as assessed by RT-PCR (n = 2 experiments). ( b ) ROCK1 (top) and ROCK2 (bottom) expression (green) in RPCs cultured on 0.5 kPa substrate (left) and on 50 kPa substrates 48 h after transfection with scramble and ROCK1 and ROCK2 LNA-GapmeRs. DAPI counterstains nuclei (white). Bars = 75 μm. ( c ) Quantification of ROCK1 and ROCK2 fluorescence intensity in RPCs cultured on substrate with 0.5 kPa stiffness and in RPCs cultured on 50 kPa substrates 48 h after transfection with scramble and ROCK1 and ROCK2 LNA-GapmeRs. At least 50 cells for each condition were analyzed. Box-and-whisker plots: line = median, box = 25–75%, whiskers = 10–90%. ( d ) Confocal images of F-actin immunodetection by phalloidin (red), paxillin (green) and nuclei with DAPI counterstain (white) in RPCs cultured on 50 kPa substrate treated with scramble and with ROCK1 and ROCK2 LNA-GapmeRs. Bars = 25 μm. ( e ) Percentage of cell viability in scramble and LNA-ROCK1 and ROCK2 treated RPCs cultured on 50 kPa substrate versus RPCs grown on 0.5 kPa substrate. Data are mean ± SE (replicate n = 4). ( f ) MSD of RPCs treated with scramble or LNA-GapmeR for ROCK1 and ROCK2 and cultured on 50 kPa stiffness (n = 45, 30, and 32 trajectories for 50 kPa + scramble, 50 kPa + LNA-ROCK1, and LNA-ROCK2, respectively). ( g ) Average speed of RPCs transfected with scramble or LNA-GapmeR for ROCK1 and ROCK2 and cultured on 50 kPa. At least thirty randomly selected RPCs were monitored over 3 h for each condition. Data are expressed as mean ± SE. * p < 0.05 using one-way ANOVA followed by Tukey’s or Bonferroni post-hoc test, as appropriate.

Article Snippet: The following antibodies were used: Anti-paxillin (ab32084, dilution 1:250, Abcam, Cambridge, UK), anti-nephrin (NMP1-30130Y17-R, dilution 1:200, Novus Biologicals Centennial, CO, USA), anti-ROCK1 (Cat# 202694T02, dilution 1:200, Sino Biological, Wayne, PA, USA), anti-ROCK2 (Cat# HPA044109, dilution 1:30, Sigma Aldrich, Milan, Italy).

Techniques: Migration, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transfection, Fluorescence, Whisker Assay, Immunodetection