Supplementary Figure S9 from Intracellular Osteopontin Promotes the Release of TNFα by Mast Cells to Restrain Neuroendocrine Prostate Cancer

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Supplementary Figure S9. Expression and silencing of putative TLR ligands in NEPC cells. A. Venn diagrams show a list of 29 genes (List 3; Supplementary Table S3) extrapolated from the intersection between genes found up-regulated in TRAMP-derived incipient NEPC (a data set generated in this study, GSE242811; List 1; Supplementary Table S3) and a list of surface TLR2/TLR4 ligands identified from a ligand-receptor pairs repository (LewisLabUCSD, ref. (Armingol et al., 2021) List 2; Table S3). B. Real Time-PCR for Cd14, Sdc1, Hspa2 and Anxa2 on lysates from T23 and ST4787 cells. Technical replicates are indicated by dots (n=2). C. Quantification of the western blot for HSPA2 and ANXA2 in T23 and ST4787 showed in Fig. 6B. D. Expression of CD14 and SDC1 in ST4787 silenced with two different siRNA specific for Cd14 (ST4787-siCD14- 1 and ST4787-siCD14-2) or Sdc1 (ST4787-siSDC1-1 and ST4787-siSDC1-2), or in scramble control cells (ST4787-scramble), evaluated by flow cytometry 72 hours post-silencing. E. HSPA2 and ANXA2 expression in ST4787 silenced with two different siRNA specific for Hspa2 (ST4787- siHSPA2-1 and ST4787- siHSPA2-2) or Anxa2 (ST4787-siANXA2-1 and ST4787-siANXA2-2), or in scramble control cells (ST4787-scramble) evaluated by western blot analysis 72 hours post- silencing. F. Quantification of E. Western blot was validated twice. G. ST4787-scramble or silenced with two different siRNA specific for Cd14 were cultured with WT or OPN−/− MCs (ratio 1:1). After 16 hours the percentage of TNF positive MCs was evaluated by intracellular flow cytometry in WT or OPN-/-MCs. Gating strategy is reported in Fig. S4D. H. Flow cytometry evaluation of CD14 on adenocarcinoma (22Rv1 human) and NEPC (TC411K murine, and NCI-H660 human) cells. All histograms depict mean ± SD of biological replicates (represented by dots). T test (B) or One-way ANOVA followed by Tukey’s multiple comparison test (G) were used for the analysis of significance between samples. P-values are reported as: *, P < 0.05; **, P <0.01; ***, P <0.001. Where P-value is not indicated, the comparison between groups is not statistically significant.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.