Determination of differential IC₅₀ (DIC₅₀) effects of JA on various TNBC cell lines and TNBC PDX-derived cell lines.

<p>WST-1 proliferation assays for 48 hours utilizing JA at various dosages ranging from 100 nM-30 μM: <b>(A)</b> MSL-subtype TNBC cell lines MDA-MB-231 (EA) vs. MDA-MB-157 (AA). <b>(B)</b> BL1-subtype TNBC cell lines HCC-38 (EA) vs. MDA-MB-468 (AA). <b>(C)</b> TNBC PDX-derived “naïve” cell line HCl-2 vs. “chemoresistant” HCl-10. The IC₅₀ of JA indicated for each cell lines determined by n = 3, in triplicate, using the two-tailed <i>t-test p</i>-values were calculated: *p < 0.05, **-p < 0.01 *** < 0.001 relative to vehicle control cells (DMSO).</p

Compounds JA arrests cells at S-phase of cell cycle and induced apoptosis in MDA-MB-231 Cells.

<p><b>A)</b> Annexin V-FITC staining was used to detect apoptosis by flow analysis in control (DMSO), staurosporine (1μM), ICG-001 (10 μM) and JA (2.5 μM) treated cells for 48 hours. Cells were counter stain with propidium iodide (PI, 1 μg/ml). We show one representative FACS plot <b>B)</b> Quantification of alive, early and late apoptotic cells in cells from panel A. <b>C)</b> qPCR for Survivin (<i>BIRC5</i>) in control cells, ICG-001 and JA treated cells. <b>(D)</b> One representative FACS plot using PI/DNA content analyzed by flow cytometry <b>(E)</b> Quantification of cell cycle phases for in G₀/G₁, S and G₂/M in both ICG-001 and JA treated cells. <b>(F)</b> qPCR for cell cycle markers <i>CDK4</i>, <i>CCND1</i>, <i>CCNE1</i>, <i>CCNA1 and CCNB1</i> from the same cells as in panel E. Statistics on three biological independent experiments with duplicates for each of the above. <i>T-test</i> was used to determine <i>p</i>-values: ***-p < 0.001, **-p < 0.01 and *p < 0.05 vs. control.</p

JA inhibits EMT-markers and prevents cell migration in wound healing assays.

<p><b>(A)</b> qPCR for <i>SLUG</i>, <i>FIBRONETIN</i>, <i>VIMENTIN</i> and <i>ZEB1</i> in ICG-001 and JA treated MDA-MB-231 cells. Results are expressed as mean ± SE, <i>n</i> = 3 *p<0.05. <b>(B)</b> The number of cells migrated into the scratched area was photographed (340) and calculated as a percentage of migration for 16 hours post treatment. Quantification of triplicates using <i>t-test</i>: ***-p < 0.001 vs. control.</p

JA inhibits expression on Wnt/β-catenin direct-target genes and degrades non-phosphorylated activated β-catenin protein levels.

<p><b>(A)</b> qPCR for <i>BIRC5</i>, <i>AXIN2</i>, <i>HMGA2</i>, <i>CNND1</i>, <i>MYC</i> and <i>PCNA</i> in ICG-001 and JA treated MDA-MB-231 cells. <b>(B)</b> Immunoblot analysis for AXIN2, HMGA2, CYCLIND1, PCNA and MYC. ACTIN serves as the loading control. <b>(C)</b> Immunoblot analysis for non-phosphorylated activated β-CATENIN (ABC), total pan-β-CATENIN and GS3Kβ^Ser9 from the same cells and extracts as in panel A and B. Quantification of triplicates using <i>t-test</i> p values are ***-p < 0.001, **-p < 0.01 and *p < 0.05 vs. control.</p