Fig 2: TNFa increases SRp40 phosphorylation through AKT: (A) Representative autoradiograph of Western blot analysis (left panel) of SRp40 phosphorylation (i.e., pSRp40) levels in hMSCSc at T = 0 h treated for 30 min with 10 or 20 ng/mL of TNFa cytokine or with vehicle (i.e., PBS). Hsp90 was used as a loading control. Bar graphs (right panel) report relative pSRp40 levels normalized on Hsp90 expression (pixel density analysis of Western blots). Data are representative of the mean ± SEM of three independent experiments. *** p = 0.001. (B) Representative Western blot of pSRp40 (left panel) on lysates from undifferentiated hMSCs treated for 24 h with conditioned medium (CM) of LPS-activated MF (THP-1) or control medium plus 0.5 mg/mL of human neutralizing antibody against TNFa or anti-IgG1 Isotype. Hsp90 was used as loading control. Bar graphs (right panel) report relative pSRp40 levels normalized on Hsp90 expression (pixel density analysis of Western blots). Data are representative of the mean ±SEM of three independent experiments. ** p = 0.01. (C) Representative immunoblotting of Akt phosphorylation (i.e., pAkt) and total Akt levels (right panel) in hMSCSc at T = 0 h treated for 30 min with 10 or 20 ng/mL of TNFa cytokine or with vehicle (i.e., PBS). Hsp90 was used as loading control. Bar graphs (right panel) report relative pAkt levels normalized on Hsp90 and total Akt expression (pixel density analysis of Western blots). Data are representative of the mean ± SEM of three independent experiments. (D) Representative autoradiograph of Western blot analysis (left panel) of pAkt and total Akt levels in undifferentiated hMScs treated for 24 h with the CM of LPS-activated MF (THP-1) or control medium plus 0.5 mg/mL of human neutralizing antibody against TNFa or anti-IgG1 Isotype. Hsp90 was used as loading control. Bar graphs (right panel) report relative pAkt levels normalized on Hsp90 and total Akt expression (pixel density analysis of Western blots). Data are representative of the mean ± SEM of at least three independent experiments. * p = 0.05 and ** p = 0.01. (E) Representative immunoblotting of pSRp40 (left panel) in undifferentiated hMSCs treated with Wortmannin or vehicle (i.e., DMSO) in combination and not with 10 or 20 ng/mL of TNFa cytokine. Hsp90 was used as loading control. Bar graphs (right panel) report relative pSRp40 levels normalized on Hsp90 expression (pixel density analysis of Western blots). Data are representative of the mean ± SEM of at least three independent experiments. * p = 0.05 and ** p = 0.01.

Fig 3: Inhibition of the phosphoinositide 3 -kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway activates cabazitaxel-induced autophagy. Proteins were extracted from cabazitaxel-treated A549 cells and the phosphorylation of mTOR and Akt was detected by western blotting. P-, phosphorylated.

Fig 4: Expression level of genes and proteins relating to cell proliferation, migration and invasion. (A) The gene expression levels detected by qRT-PCR. (B) The expression levels of corresponding proteins detected by western blotting. *P<0.05 and **P<0.01. AKT, protein kinase B; GSK-3ß, glycogen synthase kinase-3ß; GSLCs, glioma stem-like cells; mTOR, mammalian target of rapamycin; NF-?B, clear factor kappa-light-chain-enhancer of activated B cells; PGBCs, primary glioma-based cells; PI3K, phosphoinositide 3-kinase; p-STAT3, phospho-STAT3; STAT3, signal transducer and activator of transcription 3; Tp, total protein; VEGF, vascular endothelial growth factor.

Fig 5: RAW 264.7 macrophages were treated with 10 µM CAL-101, a specific inhibitor of p110d, the catalytic subunit of PI3Kd isoform, 30 min before the addition of 15 µM benznidazole (Bzl). Cells were stimulated with 10 µg/mL of LPS 30 min after Bzl treatment. After 30 minutes, relative p-P70S6K (70 kDa) and relative p-AKT (60 kDa) protein expression was determined by Western blot with a specific antibody and normalized against total P70S6K (70 kDa) and AKT (60 kDa), respectively. Results are expressed as the mean of 3 independent experiments (n=3, 5 replicates/treatment) ± SEM. ?P < 0.05 vs. Bzl-treated and LPS-stimulated cells.