1. RUNX3 depletion induces cellular senescence and inflammatory cytokine expression in cells undergoing TGFβ-mediated EMT. A, Cells were transfected with control or RUNX3 siRNA and treated with vehicle control or TGFβ for 48 hours.
RUNX3 depletion induces cellular senescence and inflammatory cytokine expression in cells undergoing TGFβ-mediated EMT. A, Cells were transfected with control or RUNX3 siRNA and treated with vehicle control or TGFβ for 48 hours. Immunofluorescence staining was performed with γH2AX antibody and Alexa Fluor-594–conjugated phalloidin. The phalloidin and γH2AX images were imaged individually and merged using Adobe Photoshop software. Merged images represent coimmunofluorescence for phalloidin and γH2AX staining. Scale bar, 50 μm. B, Experiment was performed as described in A. Scatter plot shows projected nuclear area across samples. C, Experiment was performed as described in A. Images were captured and subjected to quantitative image analysis to compute nuclear area. The average γH2AX foci/cell within cells with the nuclear area 100–250 μm2 or greater than 250 μm2 is shown. One-way ANOVA nonparametric test was used for statistical analysis. ***, P < D, Cells were treated as described in A. Samples were fixed and stained for SA-β-gal enzyme overnight at 37°C. Inset image was zoomed (200%) and is shown below. Scale bar, 50 μm. E, SA-β-gal–positive cells (blue) were scored and plotted. Quantification of SA-β-gal positivity across two independent experiments, each done as triplicates. F, Cells were transfected with RUNX3 siRNA. GFP-tagged siRNA-resistant RUNX3 or GFP-HMOX1 were expressed for 24 hours, following which, cells were sorted by flow cytometry into GFP-positive and GFP-negative populations and plated. After TGFβ exposure for 48 hours, the SA-β-gal assay was performed. G, RNA-Seq data described in Fig. 4A was subjected to pathway analysis. Gene ontology (GO) pathways enriched under the category “Molecular function” are shown. Control and RUNX3-KD cells treated with TGFβ were subjected to differential gene expression analysis, as described in Materials and Methods. Genes regulated by RUNX3 in a TGFβ-independent manner were excluded. The online tool, Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7, was used for GO analysis. Genes expressing more than 200% of control were considered upregulated, whereas those with expression less than 50% of control were considered as downregulated. List of differentially expressed genes are shown in Supplementary Table S1. H, Heatmap depicting overexpression of SASP genes. Gene expression derived from FPKM values was plotted. Biological replicates for each sample, is represented individually. I, miR-146a FPKM values derived from RNA-Seq analyses are shown. J, Experiment was performed as described in A. Samples were harvested after 48 hours of TGFβ treatment. qPCR analyses were done for the indicated genes. Graphs show mean ± SD. Asterisks represent significant differences. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant.
Vaidehi Krishnan et al. Cancer Res 2018;78:88-102
©2018 by American Association for Cancer Research