Journal: Frontiers in Oncology

Article Title: Indolyl-chalcone derivatives trigger apoptosis in cisplatin-resistant mesothelioma cells through aberrant tubulin polymerization and deregulation of microtubule-associated proteins

doi: 10.3389/fonc.2023.1190988

Figure Lengend Snippet: Role of STMN1 and CRMP2 in mediating the anti-tumor effects of CIT-026 and CIT-223. (A, C) Both MPM cell lines PXF698 (A) and PXF1752 (C) expressed high levels of STMN1 whereas significant amounts of CRMP2 were only expressed in PXF1752, determined by immunohistochemistry. Scale bar, 100 µm. (B, D) Immunoblotting with STMN1 and CRMP2 specific antibodies of PXF698 (B) and PXF1752 cells (D) treated with increasing concentrations of CIT-026 or CIT-223 for 24 h (top) or 1 µM CIT-026 or DMSO for 1 h, 4 h and 24 h (bottom). Bar graphs represent semi-quantitative analysis of phospho-protein expression relative to total protein, normalized to protein levels in DMSO treated cells. Mean ± SD of two independent experiments. (E, F) PXF698 (E) and PXF1752 cells (F) were transfected with STMN1-siRNA or non-target control (NTC)-siRNA. Knockdown of STMN1 and suppression of phospho-STMN1 (S16) induction after exposure to CIT-026 (1 µM, 24h) were confirmed by immunoblotting. Cell viability of transfected PXF698 (E) and PXF1752 cells (F) exposed to the indicated concentrations of CIT-026 for 96h and 72h, respectively, was measured by MTT assay. Mean ± SD of five replicate samples, representative of two independent experiments. Statistical significance was determined by Two-Way ANOVA and Bonferroni posttests, **P<0.01; ***P<0.001¸ ns, P>0.05.

Article Snippet: Antibodies to detect STMN1 (D1Y5A, #13655), phospho-STMN1 (Ser16, #3353), CRMP2 (D8L6V, #35672), phospho-CRMP2 (Thr514, #9397), PARP (46D11, #9532), c-Jun (60A8, #9165), phospho-c-Jun (Ser63, #91952), AKT (C67E7, #4691), phospho-AKT (Ser473, D9E, #4060), ERK1/2 (137F5, #4695), phospho-ERK1/2 (Thr202/Tyr204, #4370), RPS6 (5G10, #2217), phospho-RPS6 (Ser235/236, #4858) were from Cell Signaling (Danvers, MA, USA).

Techniques: Immunohistochemistry, Western Blot, Expressing, Transfection, Control, Knockdown, MTT Assay

Journal: Frontiers in Oncology

Article Title: Indolyl-chalcone derivatives trigger apoptosis in cisplatin-resistant mesothelioma cells through aberrant tubulin polymerization and deregulation of microtubule-associated proteins

doi: 10.3389/fonc.2023.1190988

Figure Lengend Snippet: Mechanistic scenario of the molecular action of CIT-026 and CIT-223. CIT compounds promote the assembly of aberrant microtubule fibers via direct interaction with tubulin and phosphorylation of microtubule regulatory proteins STMN1, CRMP2 and WNK1. Formation of aberrant tubulin fibers induces abnormal spindle morphology, mitotic arrest and apoptosis. Apoptosis is mediated by activation of pro-apoptotic signaling (Chk2, STMN1, JNK/c-Jun, HSP60) and inhibition of AKT/ERK, thus counteracting stress-induced pro-survival signaling (HSP60, c-Jun, CREB).

Article Snippet: Antibodies to detect STMN1 (D1Y5A, #13655), phospho-STMN1 (Ser16, #3353), CRMP2 (D8L6V, #35672), phospho-CRMP2 (Thr514, #9397), PARP (46D11, #9532), c-Jun (60A8, #9165), phospho-c-Jun (Ser63, #91952), AKT (C67E7, #4691), phospho-AKT (Ser473, D9E, #4060), ERK1/2 (137F5, #4695), phospho-ERK1/2 (Thr202/Tyr204, #4370), RPS6 (5G10, #2217), phospho-RPS6 (Ser235/236, #4858) were from Cell Signaling (Danvers, MA, USA).

Techniques: Phospho-proteomics, Activation Assay, Inhibition