Fig 2: ERα36 overexpression enhancing M4-dependent migration but not proliferation or apoptotic escape. (A) Representative western blot of a M4 kinetic treatment of the MCF-10A cell line. M4 (1 nM) stimulated ERα36 protein expression. β-Actin was used as a loading control. N = 3. (B) Quantification of MCF-10A/ERα36 cell viability by crystal violet assay after 48 h vehicle or 1 nM M4 exposure. Histogram depicts the percentage of MCF-10A/ERα36 viable cells compared to MCF-10A/Zeo cells after treatment. M4 triggered a significant 18. 4% increase of MCF-10A/ERα36 cell proliferation rate. Each bar represents mean ± SD. N = 5. * = significantly different from MCF-10A cells. aa = significantly different from MCF-10A/ERα36 vehicle treated cells (*p < 0.05; aa p < 0.01). (C) MCF-10A/Zeo or MCF-10A/ERα36 cells were exposed to 0.25 µM staurosporine (STS) for 6 h. Quantification of cleaved Caspase 3 (cCasp3), Caspase 7 (cCasp7) or PARP1(cPARP1) levels showed that ERα36 overexpression triggered a reduction of apoptotic markers. Each bar represents mean ± SD. N = 3. *p < 0.05. (D) MCF-10A cells were pretreated for 24 h with vehicle or 1 nM M4, and then exposed to 0.25 µM STS for 24 h. The percentage of apoptotic cells was assessed by TUNEL assay. Results were represented as MCF-10A/ERα36 versus MCF-10A/Zeo cells ratio. No significant additive effects of ERα36 overexpression and M4 treatment were observed concerning apoptosis escape. Each bar represents mean ± SD. N = 5. *p < 0.05. (E) A wound was performed on a confluent monolayer culture of either MCF-10A/Zeo or MCF-10A/ERα36 cells pretreated for 48 h with vehicle or 1 nM M4. Histogram depicts the percentage of wound healing for MCF-10A/ERα36 versus MCF-10A/Zeo cells after 6 h. Results indicated that M4 treatment enhanced the migratory potential of MCF-10A/ERα36 cells. Each bar represents mean ± SD. N = 3. * or ** = significantly different from MCF-10A cells. a = significantly different from MCF-10A/ERα36 vehicle treated cells (* or a: p < 0.05; **p < 0.01). ERα36, estrogen receptor alpha 36.

Fig 3: “Claudin-1-high” TNBC cells are more sensitive than “claudin-1-low” TNBC cells to chemotherapy. HCC1806, MDA-MB-231 and Hs578T cells were treated for 72 h with increasing doses of (A) 5-FU (0, 5, 10, 25, 50, 100, 200 µM), (B) PTX (0, 1, 2.5, 5, 10, 15, 20, 25 nM) and (C) DOX (0, 5, 10, 20, 30, 50, 160, 400, 800 nM). Cell viability was determined by measuring ATP level. The results are expressed as a percentage of the control cells exposed to the solvent and IC50s are specified above the graphs. Cells were treated for 72 h at different concentrations of (D) 5-FU (0, 25, 50, 100, 150, 200 µM); (E) PTX (0, 1, 2.5, 5, 18 nM); and (F) DOX (0, 10, 25, 50, 150, 400 nM). Analysis of CLDN1, cleaved apoptotic markers PARP-1 (cPARP) and caspase 7 (cCASP7) expression was performed by western blot. The GAPDH was used as an internal reference. Western Blots expression are representative of 3 to 4 independent experiments. The values represent the means ± SEM of three to four different experiments. A p-value of less than 0.05 was considered statistically significant with * p < 0.05. The uncropped blots are shown in File S1.

Fig 4: M4 stimulating resistance to apoptosis. (A) Western blot analyses of protein expression level (left) and corresponding quantifications (right) of phospho-Bad (ser136), phospho-Caspase 9 (ser196) in 24 h vehicle or 1 nM M4-treated cells. M4 treatment triggered a significant 19% and 67% increase of Bad (ser136) and Caspase 9 (ser196) phosphorylation, respectively. Each bar represents mean ± SD. N = 3. *p < 0.05. (B) MCF-10A cells were pretreated for 24 h with vehicle or 1 nM M4, and then exposed to 0.25 µM staurosporine (STS) for 6 h or not (Veh). Cleavage of PARP1, Caspase 7, and Caspase 3 were evaluated with specific antibodies. β-Actin was used as a loading control. Results are represented as M4 + STS/STS ratio. M4 treatment triggered a significant 36, 48, and 17% decrease of PARP1, Caspase 7, and Caspase 3 cleavage, respectively. Each bar represents mean ± SD. N = 4. *p < 0.05.

Fig 5: CLDN1 overexpression sensitizes Hs578T cells to PTX and DOX but not to 5-FU. (A) Hs578T cells stably transfected with CLDN1 vector (Hs578T/CLDN1) or the empty vector (Hs578T/CTRL) were treated for 72 h with (B,C) PTX at 5 nM, (D,E) DOX at 210 nM and (F,G) 5-FU at 181 µM, or with solvent (Ctrl). (A) CLDN1 expression was studied by western blot. (B,D,F) Hs578T cells were co-labeled with annexin V coupled to FITC and with propidium iodide and the living, early- and late-apoptotic cells were analyzed by flow cytometry. (C,E,G) Cleavage of apoptotic markers PARP-1 (cPARP) and caspase 7 (cCASP7) expression were analyzed by Western blot. Relative protein level expressions correspond to the protein of interest on GAPDH ratios. The values represent the means ± SEM of three to four different experiments. A p-value of less than 0.05 was considered statistically significant with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The uncropped blots are shown in File S1.