Fig. 4: PIAS2b-dsRNAi induces mitotic catastrophe at the onset of mitosis in anaplastic thyroid cancer cells. PIAS2b localizes at the mitotic spindle and binds microtubule and proteasomal proteins.

a Representative photograms of 48-h Sir-DNA stained time-lapse with ns- or PIAS2b-dsRNAi in one ATC cell line (8305C), one ATC primary culture (T-UC1), and the PDTC (papillary origin) cell line B-CPAP; (fully in Supplementary Fig. 2). Individual Cells traced for mitotic completion (white arrowheads) or mitotic catastrophe (blue and black arrowheads). b The 4 anaplastic cultures show massive mitotic catastrophe at mitotic onset (G2/M-prophase) compared to mitotic completion in B-CPAP. c Mitotic colocalization of tubulin-alpha (aTub, red) and PIAS2b (mPIAS2, green), in asynchronous 8305C. DAPI in blue. DIC, differential interference contrast. d EGFP-hPIAS2b transfected at low amounts (Supplementary Figs. 1x, 4c) followed by enhancing signal strategies, shows localization at the spindle compared to broad cytoplasmic staining in EGFP-C1. Phospho-Histone H3 (pHH3) indicates mitotic cells. EGFP immunostaining, or direct Xfect of EGFP-PIAS2b protein also localize at the spindle, but with background plasma membrane signal. e 8305C and CAL-62 cells synchronized at mitosis with double-thymidine block followed by release (DT/R). Mitotic proteins AURKA or tubulin gamma accumulate at 6 h after release. PIAS2b, detected with mPIAS2 or rPIAS2b, also accumulates at DT/R-6 h. f Immunoprecipitation of PIAS2b with mPIAS2 antibody or control isotype mouse IgG2a in DT/R-6 h synchronized 8305C extracts (called Proteomic Assay 1). Replicate pull downs were analyzed by LC-MS/MS and quantified (SWATH and Spectrum Count). Identified proteins specific for PIAS2 immuno-precipitation were enriched in the mitotic REACTOME. g The three GO pathways were microtubule/spindle cytoskeleton (green), proteasome complex (red) and prophase (violet). Underlined known SUMOylated proteins. Validated proteins in this and following figures in brilliant color. h GFP-Trap pull-downs of EGFP-PIAS2b or EGFP were analyzed by LC-MS/MS (Proteomic Assay 2). A cross-data analysis validates many mitotic proteins common to both Proteomic Assays 1 (f) +2 (h), yellow circles in g. For b n = 6 (8305C, T-UC1)-8 (CAL-62)-3 (T-UC3) and 15 (BCPAP) independent experiments; two-sided Mann–Whitney. Bar indicate means ± SEM; nd, not detected; when significant the exact p value is indicated in the figure. For c and d n = 5 independent experiments. For e and f n = 3, and for g and h Proteomic Assay 1 n = 5 and Proteomic Assay 2, n = 3 independent experiments; quantifications in Supplementary Data 3. Source data provided.