Fig 2: Detached endfeet, caused by loss of Myh10, impact cortical architecture.(A) Cartoon schematic of an RGC (green) and the local niche where the endfeet reside made up of BM (black), fibroblasts (brown), vasculature (red), CR cells (grey), and interneurons (red). Box indicates MZ region of images in B, E, G. (B) Labeling of all nuclei with DAPI (blue) in E16.5 Myh10 Ctrl and cKO brains. Boundary between pia and MZ marked by yellow dashed line. White bar represents MZ thickness measurements in D. (C) Quantification of DAPI cells in the MZ. n = 5 Ctrl, 6 cKO from 2 litters. (D) Quantification of MZ thickness. Each data point represents the average of 3 measurements per section and 3 sections per brain. n = 6 per genotype from 2 litters. (E) Staining with Calretinin (white) to label CR cells and interneurons and DAPI (blue) to mark all nuclei in E16.5 Myh10 Ctrl and cKO brains. Yellow bar represents where Calretinin thickness was measured for F. (F) Quantification of Calretinin thickness. n = 3 per genotype from 1 litter (G) P73 staining (green) of CR cell nuclei and DAPI (blue). Asterisks denote vasculature. White dotted line marks boundary between pia and MZ. (H) Quantification of P73+ cells in MZ. n = 6 per genotype from 2 litters Error bars: SD. (C, D, F, H). Student unpaired, two-tailed t test (C, D, F, H). Data points color-coded by litter (C, D, F, H). ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Scale bar: (B, E, G), 50 μm. Data underlying graphs included in S4 Data. BM, basement membrane; CR cells, Cajal–Retzius cells; MZ, marginal zone; RGC, radial glial cell.

Fig 3: MC3-indued cytotoxicity appears to be independent of the tumor suppressor, TAp73. (A) Western blot analysis reveals a strong reduction in the two major isoforms of p73, TAp73 and ΔNp73, in WT and p53−/− HCT116 cells treated with either 5 μM of MC3 or the vehicle (0.1% DMF) for 24 h. β-actin and vinculin were used as loading controls. Numbers show values obtained from densitometric analyses of the presented blots normalized to the respective loading controls (β-actin). (B) 24 h of treatment with MC3 at the indicated concentrations down-regulates the TP73 gene in the three CRC cell lines. mRNA levels were analyzed by qRT-PCR and the relative gene expression was determined using the ΔΔ Ct method where the Ct values of TP73 were normalized to those of β-actin. 0.1% DMF was used as mock. Error bars indicate the SEM of three biological replicates. (C) Knock-down of p73 in HCT116 p53−/− does not hamper MC3-induced PARP degradation as well as p21 induction. p53-deficient HCT116 cells were transfected with anti-p73 siRNA (HCT116 p53−/−p73KD) or a negative control (HCT116 p53−/− siRNA NC), thereafter were treated with 0.1% DMF or MC3 (5 μM) for 24 h. Transfection efficiency was confirmed by a clear reduction in p73 protein expression. β-actin and vinculin served as loading controls. Numbers represent densitometric analyses of the presented blots normalized to the respective loading controls (vinculin). (D) Viability of HCT116 p53−/−p73KD cells after 24 h treatment with 5 μM of MC3 is reduced in a similar manner to HCT116 p53−/−siRNA NC as well as the parental cell line, measured by MTT assay. Percentage cell viability was determined by normalizing the results of MC3-treated cells to those of vehicle (0.1% DMF)-treated ones. Data represent mean ± SD from two independent experiments, each was done in triplicates.

Fig 4: TAp73 induces the expression of epithelial genes and SMAD4. (A) PANC-1 cells were transiently transfected twice with 50 nM each of p73 siRNA or an irrelevant control (Co) siRNA and 48 h after transfection subjected to qPCR for ECAD, GRHL2 and SMAD4, as well as GAPDH as normalization control. Data represent the normalized mean ± SD of three assays. (B) As in (A), except that cells were subjected to immunoblotting for ECAD (left-hand panel) or SMAD4 (right-hand panel), and HSP90 as loading control. Successful knockdown of p73 was verified by immunoblot analysis (see Figure S1). (C) Immunoblot analysis show successful overexpression of TAp73α and TAp73β in PANC-1 cells transfected with either empty vector (V) or expression vectors for the two p73 isoforms and incubated with a p73 antibody. The bands of the endogenous p73 protein are not visible here due to the short exposure time of the blot. (D) PANC-1 cells were transiently transfected with empty vector (V) or expression vectors for TAp73α or TAp73β, and processed for qPCR analysis of ECAD, GRHL2 and SMAD4, and GAPDH as a control. Data represent the normalized mean ± SD of three experiments. The asterisks (*) in (A,D) denote a significant difference relative to the control (p < 0.05, Wilcoxon test); ns, non-significant.

Fig 5: TAp73 interferes with basal and TGF-β1-induced ERK activation. (A) PANC-1 cells were transiently transfected with 50 nM each of p73 siRNA or an irrelevant control (Co) siRNA and were either left untreated (left-hand blot) or were treated 48 h after transfection for various times (as indicated) with 5 ng/mL TGF-β1. Subsequently, cells were subjected to immunoblotting of pERK1/2 and either total ERK1/2 (left-hand blot) or GAPDH (right-hand blot) to control for equal loading. Since we observed that within the 2 h observation period, neither GAPDH nor HSP90 expression was affected by modulation of TAp73 expression, these housekeeping proteins rather than total ERK1/2 were used for normalization of pERK1 and pERK2 levels. (B) The same as in (A), except that HPAFII cells were used. Successful knockdown of TAp73 in (A) was verified by immunoblotting (Figure S1) and in (B) by qPCR. The graphs below the blots show results from densitometry-based signal quantification of three experiments (means ± SD, n = 3). The asterisks (*) indicate significance (p < 0.05, Wilcoxon test) relative to the respective Co siRNA sample.