Fig 2: TNFa and TGFß1 cooperate in inducing the protein expression levels of CCL2, CXCL8 and Cox-2, and induce the activation of the NF-?B and Smad3 transcription factors, respectively, in MSCs. Human BM-derived MSCs were stimulated with vehicle (“--”), TNFa (50 ng/ml), TGFß1 (10 ng/ml), or TNFa + TGFß1 (same concentrations). Ta + Tß = TNFa + TGFß1. (A) Protein expression levels of CCL2, CXCL8 and Cox-2. Following 24 h of stimulation (FBS-free medium for CCL2/CXCL8 and 0.5% FBS-containing medium for Cox-2) the extracellular expression of CCL2 [(A1); see also Figure S3 in Supplementary Material] and CXCL8 (A2) was determined by ELISA in cell supernatants, in the linear range of absorbance. ***p = 0.001 compared to vehicle-treated cells. (A3) Cox-2 expression levels were determined by WB; GAPDH was used as a loading control. (B) Following 10 and 30 min of stimulation (FBS-free medium), the phosphorylation levels of p65 (B1) and Smad3 (B2) were determined by WB; GAPDH was used as a loading control. In all panels, the findings are representatives of n = 3 independent experiments, performed with MSCs of two to three different donors, which have shown similar results.

Fig 3: Analyses of human breast tumors demonstrate coordinated expression of CCL2, CXCL8 and Cox-2, and higher levels of their expression in tumors enriched with both TNFa and TGFß1. Patient data analysis was performed with the TCGA dataset of human breast cancer, including 1,215 patient samples. (A) Associations between CCL2 (A1), CXCL8 (A2), Cox-2 (A3) expression and expression levels of TNFa and TGFß1 in the tumors, performed by quartile analysis, as described in Section “Materials and Methods”. The black dots above the plots are outliers that are outside of the whiskers. Statistical analysis was performed by two-tailed Mann–Whitney test. In panel A1, ***p < 0.001 for CCL2 expression levels in the TNFa-high + TGFß1-high patient group, compared to all other patient groups. In panel A2, ***p < 0.001 for CXCL8 expression levels in the TNFa-high + TGFß1-high patient group, compared to all other patient groups. In panel A3, ***p < 0.001 for Cox-2 expression levels in the TNFa-high + TGFß1-high patient group, compared to the TNFa-low + TGFß1-low group, **p < 0.01 compared to the TNFa-high + TGFß1-low group and *p < 0.05 compared to the TNFa-low + TGFß1-high group. (B) Associations between CCL2, CXCL8 and Cox-2 expression levels in the patient cohort. The correlation coefficient (r) and statistical significance between two mRNAs in each graph were determined by Spearman correlation analysis. rsem, RNA-Seq by Expectation Maximization. Similar analysis performed with the METABRIC dataset of patient samples is demonstrated in Figure S2 in Supplementary Material.

Fig 4: TAK1 plays divergent roles in regulating TNFα + TGFβ1 cooperatively-induced CCL2/CXCL8 and Cox-2 expression in MSCs. (A) Human BM-derived MSCs were stimulated with vehicle (“--”), TNFα (50 ng/ml), TGFβ1 (10 ng/ml), or TNFα + TGFβ1 (same concentrations) for 10 min (FBS-free medium). Tα + Tβ = TNFα + TGFβ1. TAK1 expression was determined by WB; GAPDH was used as a loading control. The findings are representatives of n = 3 independent experiments, performed with MSCs of three different donors, which have shown similar results. (B,C) The effects of TAK1 downregulation on MSC signaling and functions. siRNA concentrations were determined by preliminary titration analysis (data not shown). (B) Signaling: human BM-derived MSCs were transiently transfected with control siRNA (“CTRL”, 50 nM) or with siRNA to TAK1 (50 nM; efficacy of TAK1 downregulation is demonstrated in Figure S6A1 in Supplementary Material). Following siRNA transfection, the cells were stimulated with vehicle (“--”) or with TNFα (50 ng/ml) + TGFβ1 (10 ng/ml) for 10 min (0.5% FBS-containing medium). Tα + Tβ = TNFα + TGFβ1. (B1) The expression levels of IκBα were determined by WB; GADPH was used as loading control. (B2) p65 and Smad3 activation levels were determined as described in Figure 5. p38 activation levels are demonstrated in Figure S6A2 in Supplementary Material. (C) Function: human BM-derived MSCs were transiently transfected with control siRNA (“CTRL”, 50 nM) or with siRNA to TAK1 (50 nM; efficacies of TAK1 downregulation are demonstrated in Figures S6B1,B2 in Supplementary Material). Following siRNA transfection, the cells were stimulated with vehicle (“--”) or with TNFα (50 ng/ml) + TGFβ1 (10 ng/ml) for 24 h (0.5% FBS-containing medium). Tα + Tβ = TNFα + TGFβ1. The impact of TAK1 siRNA on the expression of CCL2 (C1), CXCL8 (C2) and Cox-2 (C3) was determined as described in Figure 5. NS, not significant. In all panels, the findings are representatives of n = 3 independent experiments, performed with MSCs of two different donors, which have shown similar results. Similar findings, obtained in MSCs from another donor, are demonstrated in Figure S7 in Supplementary Material (each panel with its corresponding validation of downregulation efficacy).