Figure 6

Nickel and zinc induce non-apoptotic cell death whereas mutant p53s and C19MC miRNAs induce transformation of Hep3B cells with depolarized mitochondria. (a,b) Nickel and zinc co-operatively induce DNA condensation in Hep3B cells that are stably overexpressed with C19MC miRNAs at 24 h of treatment (Panel-a). The mitotic DNA condensation (arrow heads) was carefully excluded from cell death related condensation (white and hollow arrows). Note the diffuse cytoplasmic DNA in combination treatment (*) [Panel-b]. (c) Neither nickel nor zinc or their combination induced DNA fragmentation at 24 h of treatment. Equal quantities of gDNA were run on 2% agarose gel. (d,e) Transient transfections of mutant p53s induce transformation (arrows) of Hep3B cells with mitochondrial depolarization at 48 h, as evaluated by JC1 and Hoechst-33342 staining. The microscopy images were pseudo-colored as indicated. Green corresponds to JC1 staining of depolarized mitochondria (MOMP). Red corresponds to DNA in DAPI channel. (f) REST immunofluorescence (Red) analysis of miR-520G (GFP) stable Hep3B cells showing cellular transformation which is surrounded by Hoechst-33342 positive (Blue) nuclei. Note: miRs-519D and 526B also stimulate similar transformation (Data not shown). (g) Evaluation of 30 cell death regulators that shown differential expression between p53TCTI dataset (Fig. 2c) in RNA-seq data of Hep3B cells stably transfected with C19MC miRNAs (Heatmap) and potential mitochondrial depolarization alteration that may explain lack of DNA fragmentation in Fig. 6 panel-c. Right panel: Schematic representing the differences in endonuclease-G release between p53TC and p53TI cells. The DNA was created using Lightwave-3D (https://www.newtek.com/lightwave/) v11.6.3. Rest of the image was created and composited in Adobe Photoshop CS5 (https://www.adobe.com/products/photoshopfamily/) v 12.0 × 64.