Fig. 3: PIAS2b-dsRNAi blocks growth of patient-derived primary cultures of ATC but not those derived from differentiated thyroid carcinomas, thyroid follicular nodular disease/multinodular goiter, or normal thyroid.

a Expression of PIAS2 mRNA detected by RT-qPCR for detection of all isoforms (global PIAS2), PIAS2b or PIAS2a isoforms in primary cultures established from thyroid tissues. Our protocol h7H obtained cultures with >95% follicular epithelium. b–f Transfection of PIAS2b-dsRNAi (orange bars) significantly and specifically reduces PIAS2b expression. For b, mRNA expression of beta and alpha PIAS2 isoforms in primary cultures from normal thyroid (T-NT2) or anaplastic thyroid carcinoma (T-UC) transfected with non-target sequence (ns-dsRNAi) or PIAS2b-dsRNAi. Western blot of treated extracts confirmed downregulation of PIAS2b in primary cultures from normal thyroid (T-NT) (c), multinodular goiter (T-MNG) (d), differentiated papillary thyroid carcinoma (T-PC) (e), and ATC (T-UC, five cultures from three patients) (f). g, h Cell growth of the primary cultures in the presence of PIAS2b-dsRNAi (orange bars) expressed as percentage of the ns-dsRNAi control (100%, white bar for each culture). Blue striped bars, indicate the percentage of transfection. Genotype and TERT expression for the most common events in thyroid cancer are shown below. Some of those cultures were established from single patient surgery paired surpluses, obtained from different pathology as assessed by the clinical pathologist (Supplementary Data 2): T-NT39 and T-M19; T-NT52 and T-PC48; T-MNG100 and T-PC46; T-MNG197 and T-PC64, T-PC65; T-MNG94, and T-UC1, T-UC2; T-UC7 and T-UC8. Numbers refers to the code number for each within the pathological group. i–m Time-course experiments in some primary cultures showed no effect on cell growth of PIAS2b-dsRNAi in normal/ benign cultures (i, j) but confirmed progressive cell loss in ATC cultures from the three patients (k–m). a n included in the section, two-sided Kruskal–Wallis; (b) n = 3–4 (T-NT2), n = 5–6 (T-UC1, T-UC2, T-UC7, T-UC8), n = 6–3 (T-UC3) independent experiments, one-sided Mann–Whitney; g, h n of independent experiments: n = 3 (T-NT47, T-MNG94, T-UC3), n = 4 (all the other cultures), n = 6 (T-UC1, T-UC2, T-UC7, T-UC8), n = 8 (T-MNG100), n = 10 (T-NT2); i n = 4, j–l n = 3, k, m n = 6. b, g, h Two-sided Mann–Whitney; i two-sided one-way ANOVA with repeated measures and Dunnett’s multiple correction; j two-sided one-way ANOVA with repeated measures and Bonferroni multiple correction; k–m two-sided one-way ANOVA with repeated measures, and Tukey’s multiple comparison test. Bar indicate means ± SEM; when significant the exact p value is indicated in the figure. ns non-significant. Source data are provided as a Source Data file.