Fig. 3: BA inhibits H3S28 phosphorylation dependent on 14-3-3ζ. | British Journal of Cancer

Fig. 3: BA inhibits H3S28 phosphorylation dependent on 14-3-3ζ.

From: Benzaldehyde suppresses epithelial-mesenchymal plasticity and overcomes treatment resistance in cancer by targeting the interaction of 14-3-3ζ with H3S28ph

Fig. 3

a Immunoblot analysis of histone H3, H3S28ph, and H3S10ph in BxPC-3 cells treated with BA at 100 or 500 µM or with DMSO vehicle for the indicated times. b Pull-down assay with GST-tagged 14-3-3ζ (or GST) and high-salt extracts of BxPC-3 cells that had been treated with DMSO (D) or 1000 µM BA (B) for 100 min. The pull-down material as well as the original extracts (Input) were subjected to immunoblot analysis with antibodies to H3S28ph, 14-3-3ζ, and GST. The lane labeled HeLa acid extract (PTX: Paclitaxel treated) indicates positive control of H3S28ph, Recomb. GST-14-3-3ζ / D and Recomb. GST-14-3-3ζ / B indicate recombinant GST-14-3-3ζ with DMSO, and recombinant GST-14-3-3ζ with BA, respectively. c CellTiter-Glo assay for parental (P-) or osimertinib-resistant (O-) A549 cells expressing WT or S28A mutant forms of H3 and treated with the indicated concentrations of BA for 48 h. Data are means ± SD from n = 3 independent experiments. **p < 0.01, ***p < 0.001 for O-A549-WT versus O-A549-S28A; #p < 0.05, ##p < 0.01, ###p < 0.001 for O-A549-WT versus P-A549-WT (two-tailed Student’s t test). Immunoblot analysis of H3S28ph and 14-3-3ζ in parental (P-) and radiation-resistant (R-) PANC1 (d) or BxPC-3 (e) cells treated with DMSO (D) or 1000 µM BA (B) for the indicated times. f Immunoblot analysis of Axl in BxPC-3 cells treated with BA at 100 or 500 µM or with DMSO vehicle for the indicated times. g Immunoblot analysis of the indicated proteins in A549 cells infected with lentiviruses encoding shRNAs (sh1 or sh2) specific for 14-3-3ζ or with the empty virus (shCtrl) and then treated with DMSO (D) or 500 µM BA (B) for the indicated times. h Schematic illustration of 14-3-3 proteins and their many client proteins regulated by BA related to signaling pathways activated in cancer. i CellTiter-Glo assay for 14-3-3ζ–depleted A549 cells as in (g) treated with the indicated concentrations of BA for 48 h. Data are means ± SD from n = 3 independent experiments. ***p < 0.001 for shCtrl versus 14-3-3ζ sh1, ###p < 0.001 for shCtrl versus 14-3-3ζ sh2.

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