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[Preprint]. 2023 Sep 14:2023.09.12.557273. [Version 1] doi: 10.1101/2023.09.12.557273

Figure 2. ICD-related factors are lost in T-DM1 resistance upon TACC3 overexpression, TACC3 correlates with clinical T-DM1 resistance, and its inhibition overcomes T-DM1 resistance and restores ICD markers in vitro.

Figure 2.

A Western blot analysis of mitotic arrest, apoptosis, and ICD markers in SK-BR-3 WT and T-DM1 resistant (T-DM1R) cells treated with 0.05 µg/mL T-DM1 in a time-dependent manner. B The log fold change of the mitotic genes differentially expressed only in BT-474, SK-BR-3 or both T-DM1R cells compared to WT counterparts in RNA-seq analysis. C Western blot analysis of TACC3 protein expression in BT-474 and SK-BR-3 WT vs. T-DM1R cells. Actin is used as a loading control. D Bar graphs showing relative protein expression of TACC3 in the T-DM1-treated tumors of responder vs. non-responder patients before and after treatment (n=6–10). E Representative TACC3 IHC and H&E staining in the tumor tissues of patients from D. Scale bar=100 µm. F Percent growth inhibition in SK-BR-3 T-DM1R cells transfected with siTACC3 and treated with 0.03 µM T-DM1 (n=4–6). G Percent growth inhibition in SK-BR-3 T-DM1R cells treated with T-DM1 alone or in combination with 1 µM TACC3 inhibitor (BO-264) (n=4–6). H Western blot analysis of mitotic arrest, apoptosis, and ICD markers in BT-474 T-DM1R cells transfected with siTACC3 and treated with T-DM1. Actin is used as a loading control. I Western blot analysis of mitotic arrest, apoptosis, and ICD markers in SK-BR-3 T-DM1R cells treated with T-DM1 alone or in combination with BO-264. Actin is used as a loading control. J Relative ATP release from SK-BR-3 T-DM1R cells treated with T-DM1 alone or in combination with BO-264 (n=3, 4). K Relative HMGB1 release from SK-BR-3 T-DM1R cells treated with T-DM1 alone or in combination with BO-264 (n=3). L Percent growth inhibition in SK-BR-3 WT cells overexpressing TACC3 and treated with T-DM1 (n=3). M Western blot analysis of mitotic arrest, apoptosis and ICD markers in SK-BR-3 WT cells overexpressing TACC3 and treated with T-DM1. Actin is used as a loading control. N Relative ATP release from SK-BR-3 WT cells overexpressing TACC3 and treated with T-DM1 (n=3). Data correspond to mean values ± standard deviation (SD). Significance for D was calculated with one way Wilcoxon signed-rank test. P-values for other subfigures were calculated with the unpaired, two-tailed Student’s t test. *, P<0.05; **, P<0.01.