Fig. 1: ASD2Δ-Sh3-overexpression reduces ROCK activation in tubular cells and fibroblasts.
From: Design of precision therapeutics for a CKD risk allele by targeting Shroom3-Rock interaction
a The conserved domains of Shroom3 are shown (PDZ, ASD1, ASD2 domains and the Fyn-binding site). b Representative immunoblots of total cell lysates of HEK-293T overexpressing PDZΔ-Sh3, ASD1Δ-Sh3 or ASD2Δ-Sh3, which were immunoprecipitated with anti-V5 and probed for SHROOM3/ ROCK1. Total lysates were probed with anti-V5-tag or ACTB (one of 2 replicates). c Representative immunoblots from Dmso/HF-treated IMCD cells overexpressing GFP, WT-Sh3, and ASD2Δ-Sh3 probed for V5(Shroom3)/pMypt1/Mypt1/Rock1/Actb, and (d) relative density of pMypt1:Actb and Rock1 (n = 6 biological replicates; Unpaired T tests; ASD2Δ-Sh3 vs. GFP (p < 0.001), ASD2Δ-Sh3 vs. WT-Sh3 (p = 0.003)). e Representative phase contrast micrographs (4x) of scratches at 0 h and 16 h of IMCD cells over-expressing WT-Sh3/ ASD2Δ-Sh3 with (f) the corresponding bar graph quantifying the migrated area per field of ASD2Δ-Sh3 IMCD at 16 h (normalized to WT-Sh3 fields) obtained from 3 fields/well, & 3 wells/biological replicate (Mann-Whitney p < 0.0001). g Bar graph representing the relative mRNA expressions of pro-fibrotic/Wnt signaling transcripts, inflammatory/injury- and tubular homeostasis- genes in WT-Sh3- vs ASD2Δ-Sh3 overexpressing IMCD cells (n = 3 biological replicates; One-way ANOVA Tukey’s post test p-values are in the source data file). h Bar graph representing the relative mRNA expression of markers of profibrotic genes in 3T3 cells (Unpaired T-test p-values for Col1a1(p = 0.0017), Fn1(p = 0.0031), Col6a1 (p = 0.009) (n = 4 biological replicates), and for Serpine1(p = 0.13), Mmp2 (p = 0.12), respectively (n = 3 biological replicates). i Dot-plot shows BrdU incorporation per cell (as % of GFP-control) in 3T3 fibroblasts overexpressing WT-Sh3- vs ASD2Δ-Sh3/GFP-control with HF (hollow symbols) and without HF (solid symbols) (n = 5 biological replicates; One-way ANOVA with post-hoc Tukey’s test p-values shown in source data file). [Line and whiskers indicate mean ± SEM; Two-tailed p-values were used and denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; HF = Hydroxy Fasudil].
