Fig. 3: IAP inhibition promotes TAM-MG proinflammatory phenotype.

A Representative experiment of 3 independent experiments of IAP expression levels analyzed by Western blotting after 72 h of vehicle or SMg treatment in C8B4 microglia cell line. B Quantification of GL261-DsRed spheroids area upon vehicle (n = 10) and SMg treatment (n = 9) and in the presence or not of the C8B4 cells (n = 10). Statistical analyses were performed by using ANOVA test and ANOVA post-hoc Tukey test; alpha = 0.05, bilateral p-value: ***p < 0.0005. C Concentrations of CD14 and CCL17/TARC quantified in the supernatant of C8B4 and GL261-DsRed co-cultures by ELISA assay after 72 h of vehicle or SMg treatments (n = 6 independent experiments). Statistical analyses were performed by using Mann–Whitney test; alpha = 0.05, bilateral p-value: **p < 0.005. D Quantification of GL261-DsRed spheroids area in the presence of the C8B4 cells expressing ML-IAP (siCTRL) or down-expressing ML-IAP (siML-IAP) (n = 10) after 72 h of treatment vehicle (n = 10) and SMg treatment (n = 9). Data from three independent experiments. Statistical analyses were performed by using ANOVA test and ANOVA post-hoc Tukey test; alpha = 0.05, bilateral p-value: *p < 0.05 ; **p < 0.005 ; ****p < 0.0001. E Quantification of GL261-DsRed spheroid area in the presence of C8B4 cells treated with vehicle and SMg for 72 h. C8B4 cells were pre-treated for 24 h with ZVAD (vehicle n = 23, SMg n = 23) and TNFαi (vehicle n = 27, SMg n = 23) or without pre-treatment (vehicle n = 28, SMg n = 25). Statistical analyses were performed by using ANOVA test and ANOVA post-hoc Tukey test; alpha = 0.05, bilateral p-value: ***p < 0.0005. F Representative experiment (n = 2) of IAP expression levels analyzed by western blotting after 72 h of vehicle or SMg treatment in C8B4 microglia cell line. C8B4 cells were pre-treated for 24 h with ZVAD and TNFαi. G Representative experiment (n = 2) of expression levels of p65, phospho-p65, IκBα, phospho IκB, caspase-3, cleaved caspase-3 analyzed by Western blotting in C8B4 microglia cell line after 24 h of vehicle or SMg treatment. H Representative experiment out of 3 experiments of expression levels of CD206 and iNOS analyzed by Western blotting after 24 h of vehicle or SMg treatment. C8B4 cells were pre-treated for 24 h with ZVAD and TNFαi. Expression level of actin β served as loading control. I iNOS/CD206 ratio in C8B4 microglia cell line after 24 h of vehicle or SMg treatment. C8B4 cells were pre-treated for 24 h with ZVAD and TNFαi. Quantification was performed from 3 independent experiments using ImageJ software and data presented were normalized to actin β expression. iNOS/CD206 ratio fold changes were normalized on vehicle condition. B–E, I Bar graphs represent mean ± s.e.m.