Fig 2: Overexpression of the Hippo pathway component KIBRA/WWC1 leads to an increased level of apoptosis in podocytes but not in HEK293T cells. (a and b) Induced KIBRA/WWC1 expression in podocytes (a), but not in HEK293T cells (b) leads to increased apoptosis levels. Lysates of cells induced by treatment with doxycycline (125 ng/ml) for the indicated times were used to detect endogenous Caspase 3, Caspase 7, PARP, and their cleaved versions cleaved Caspase 3 and 7, and cleaved PARP, which are well-established markers for apoptosis. β-tubulin served as loading control. Induced KIBRA/WWC1 overexpression caused the increase in levels of apoptosis markers cleaved Caspase 3, cleaved Caspase 7 and cleaved PARP, accompanied by the stepwise decrease of the non-cleaved version of these proteins. The decreased β-tubulin signal illustrates the loss of the total amount of podocytes in which the Hippo signaling was reactivated (via KIBRA/WWC1 overexpression) for 48 h or 56 h. By contrast in HEK293T cells, the expression of the apoptotic marker and β-tubulin was stable for the entire measurement. (c) Immunofluorescence of podocytes analyzed in a show a nuclear cleaved PARP (red) staining after induction of KIBRA/WWC1 expression (nuclear staining: DAPI). Scale bars represent 10 μm. (d) The reactivation of Hippo signaling via induced KIBRA/WWC1 expression could also be confirmed in differentiated podocytes and again led to an accumulation of apoptotic marker proteins. (e) The statistic evaluation of the apoptotic marker proteins. (mean and S.D., t-test, *P<0.05)

Fig 3: Loss of KCC2 in vivo increases the levels of proapoptotic markers.A, immunoblots of cortical/hippocampal brain lysates harvested 7, 14, and 21 days after viral infection with AAV-GFP or AAV-Cre probed for the total and cleaved levels of the extrinsic proapoptotic marker caspase 8, the intrinsic proapoptotic markers caspase 3 and caspase 7, the DNA damage marker PARP, KCC2, and β-actin. B, the bar chart shows the quantification of the levels of proapoptotic markers cleaved caspase 8, cleaved caspase 3, and cleaved PARP, with significantly changed expression marked (∗p < 0.05, n = 3 replicates).

Fig 4: Mouse PHLDA1 can induce caspase-7 (but not caspase-3) activation and detachment of cells. (a) Mouse NIH3T3 cells were transiently transfected with vectors coding for indicated fusion proteins and protein extracts were analyzed by western blot. NT—not transfected cells, TR—transfection reagent only. ACTB and HSPA8 were used as loading controls. Active caspase-3 and caspase-7 were quantitated based on densitometry of replicated blots and results are shown in corresponding charts; asterisks indicate statistically significant (* p < 0.05) differences against the EGFP-transfected cells. (b) An example of the live-cell imaging of NIH3T3 cells transiently transfected with the PMAIP1/EGFP construct showing the apoptotic membrane blebbing in a cell overexpressing PMAIP1, while control cells (transfected with the EGFP vector only, right photo) were not affected. (c) An example of the live-cell imaging of NIH3T3 cells transiently transfected with vectors coding for PHLDA1/EGFP, EGFP/PHLDA1 or EGFP showing detachment of cells overexpressing PHLDA1. Images were recorded in EGFP (green) and brightfield channels for two days. The observation period shown for each vector is given above the panels. The relative time of acquisition is displayed in the upper right corner of each image.

Fig 5: TMZ − POH induces cell death via mitochondrial pathway.(A) A549, H460 and H520 cells were treated with 100 μM TMZ, POH, TMZ + POH, TMZ − POH or DMSO respectively for 48 h and subjected to apoptosis assay using Annexin-V &PI staining. Three independent experiments were performed and the results statistically analyzed as means ± SD (B). (C) Above cells were treated with 100 μM TMZ − POH for 48 h with or without presence of z-Vad-Fmk (Z-VAD), and cell viability was detected by MTT assay. Two independent experiments were performed. (D) Lysates from drug-treated A549 and H520 cells (DMSO, TMZ, POH, TMZ + POH and TMZ − POH) were subjected to western blot using following antibodies against PARP (full and cleaved PARP), cleaved CASP9, cleaved CASP7, pro-CASP3, cleaved CASP3, Cytochrome C (CYCS) and ACTB. cl = cleaved; ns = no significance. The results shown are means ± SD; ***p < 0.001.