Fig. 7: HSF2-dependent dysregulated stress response and neurodevelopmental gene expression in cells from RSTS patients.
From: CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder
a Quantification of the percentage of cells containing nSBs (nuclear HSF1-positive dots) in HD1, HD2, RSTS P2EP300, and RSTS P1CBP hPSFs transfected with HSF2 3KQ-Myc or GFP, and subjected or not to HS (1 h at 43 °C). HSF2 3KQ restores the induction of HS-induced nSBs in RSTS hPSFs. Transfection rate efficiencies: 16% for HD PSFs, 11% for RSTS hPSFs (n = 4, 100–200 cells per experiment). Error bars, mean ± SEM; *p < 0.05. Significance was calculated by two-sided multicomparison Friedmann Test. Representative immunofluorescence of RSTS hPSFs transfected with HSF2 3KQ-Myc upon HS showing nSBs identified with HSF1 (red) in transfected cells (Myc, green). Scale bar: 10 µm. See Supplementary Figs. 7 and 8a. b Representative immunoblots of protein extracts from HD1, HD2, RSTS P1CBP, and RSTS P2EP300 hPSFs showing reduced expression of N-cadherin and HSP110 levels in RSTS, compared to HD hPSFs. Quantification of N-cadherin and HSP110 levels in immunoblots, normalized to actin (n = 3). Error bars, mean ± s.d. c Representative Immunofluorescence of hPSFs at cell–cell junctions (white dotted rectangles) showing that N-cadherin (green) is reduced in RSTS, compared to HD. Yellow rectangles, magnified areas. Scale bar: 20 µm. d Representative immunoblots of protein extracts from HD2 and RSTS P1CBP hPSFs treated by vehicle (0 nM) or BTZ (5 or 10 nM) for 22 h, showing increased HSF2 protein levels by subthreshold doses of BTZ, as well as restoration of HSP110 and N-cadherin levels in RSTS, compared to HD cells. *, endogenous HSF2 before treatment; short and long, different exposure times. Quantification of N-cadherin and HSP110 levels in immunoblots, normalized to actin (n = 2). Error bar, SEM. e Representative immunoblots of HDIMR90 and RSTS P2EP300 iNPCs, showing the reduction of levels of HSF2 and its targets in RSTSEP300, compared to HD iNPCs and hCOs. Quantification of the levels of HSF2, N-cadherin (N-cadh), and HSP110, detected in immunoblots, normalized to actin (n = 3). Error bars, mean ± SEM. f Representative immunofluorescence of HDIMR90 and RSTS P2EP300 iNPCs stained with N-cadherin (green) and HSF2 (purple). g. Same as in e but with D24(±1) hCOs. (n = 3). Error bars, mean ± SEM. Source data are provided as a Source Data file.
