Figure 6. TRPA1 Mediates Oxidative-Stress Defense through Upregulation of Ca²⁺-Dependent Anti-Apoptotic Signaling Pathways.

(A) Heatmap of significantly changed phosphorylated proteins (*p < 0.05) between TRPA1- and vector-transduced MCF-10A cells in response to H₂O₂ or matrix deprivation (detached). Lysates of cells treated with 100 μM H₂O₂ for 24 hr or cultured in suspension for 24 hr were analyzed by RPPA and all signals from all samples were normalized to vector control.

(B) Heatmap of BCL-2 family proteins in TRPA1-transduced MCF-10A cells treated as in (A). *p < 0.05 comparing TRPA1 and vector.

(C) Immunoblot of lysates from the indicated MCF-10A cells treated with 100 μM H₂O₂ for 6 hr or 12 hr or cultured in suspension for 24 hr. Dotted lines indicate the cropping lines, and uncropped blots shown in Figure S5E.

(D) Ras activity in the indicated MCF-10A cells treated with 100 μM H₂O₂ for 0.5 hr or 2 hr or cultured in suspension for 24 hr, as determined by Ras-GTP pull-down assay. Uncropped blots shown in Figure S5F.

(E) Heatmap of significantly changed phosphorylated proteins (*p < 0.05) between shTRPA1 (#1 and #2)- and shGFP-transduced HCC1569 cells. Cells were treated with 10 μM H₂O₂ for 24 hr. Samples normalized to shGFP.

(F) Heatmap of BCL-2 family proteins in shTRPA1-transduced HCC1569 cells treated as in (E).

(G) Heatmap of significantly changed phosphorylated proteins (*p < 0.05), either between shTRPA1 (#1 and #2)- and shGFP-transduced HCC1569 cells or between AP-18- and its vehicle-treated H1792 cells. Cells were treated with 10 μM carboplatin in the presence or absence of 10 μM AP-18 for 24 hr. Samples normalized to shGFP for HCC1569 cells and AP-18 (–) for H1792 cells.

(H) Heatmap of BCL-2 family proteins in HCC1569 and H1792 cells treated as in (G).

(I) Ras activity in the indicated HCC1569 cells treated with 10 μM H₂O₂ for 2 hr. Normalized densitometric values are shown below each band.

(J and K) Live cell numbers relative to H₂O₂-untreated cells in the indicated MCF-10A cells treated with 70 μM H₂O₂ (J) or 100 μM H₂O₂ (K) in the presence or absence of 1 μM W-7, 10 μM TFP, 1 nM PD325901, 100 nM Torin1, 10 μM KN-93, or 3 μM A-1210477 for 72 hr. **p < 0.01 and ***p < 0.001 (one-way ANOVA).

(L and M) Live cell numbers relative to H₂O₂-untreated (L) or carboplatin-untreated cells (M) in the indicated HCC1569 cells treated with 8 μM H₂O₂ (L) or 10 μM carboplatin (M) in the presence or absence of 1 μM W-7, 10 nM PD325901, 100 nM Torin1, or 3 μM A-1210477 for 96 hr (L) or 72 hr (M). Data for control in (M) are from Figure 4L, as these samples were analyzed concurrently. *p < 0.05 and ***p < 0.001 (one-way ANOVA).

(N) Representative images of day-13 HCC1569 (shGFP) spheroids treated with or without 3 μM A-1210477. Spheroids were stained with DAPI. Scale bar, 100 μm.

(O) Pie chart of clearance levels of Day-13 HCC1569 (shGFP) spheroids treated with or without 3 μM A-1210477 (n = 44 for control and n = 37 for A-1210477: sum of two independent experiments).

(P) Model of the relevant TRPA1-regulated signaling pathways that contribute to oxidative-stress defense in cancer cells.

(Q) Immunoblot of lysates from the indicated HCC1569 or EL-12-58 tumors that were resected from mice in Figure 5A for HCC1569 tumors or Figure 5D for EL-12-58 tumors at the end point.

In (J–M), data are shown as means ± SD from the sum of three independent experiments performed in duplicate. See also Figures S6 and S7.