Additional file 1 of Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

Additional file 1: Supplementary Figure S1. Elevated cytosolic Ca2+ activates MEF2A through P38 phosphorylation and facilitates PINCH expression. (A) Neurons untreated or exposed to Tat or TNFα for 48 h were loaded with Fluo-4 AM (5 μM) to measure cytosolic Ca2+ levels. Quantification of Fluo-4 fluorescence at baseline levels. Three independent experiments were performed. Each dot represents mean fluorescence of ~ 10 cells/field and 5 fields/experiment were quantified. (B) Scr siRNA and Cav3.1 KD human neurons untreated or exposed to Tat or TNFα for 48 h were loaded with Fluo-4 AM (5 μM) to measure cytosolic Ca2+ levels. Fluo-4 fluorescence quantified as in (A). (C) Representative Western blots for lysates from Scr siRNA and Cav3.1 KD neurons untreated or exposed to Tat or TNFα for 48 h and probed with antibodies against Cav3.1, phospho-P38, P38, phospho-MEF2A, MEF2A, PINCH and GAPDH. (D-G) Quantification of relative protein abundance of Cav3.1 (D), phospho-P38 (E), phospho-MEF2A (F) and PINCH (G) from (C). (H) ChIP-assay was performed in Scr siRNA and Cav3.1 KD neurons untreated or exposed to Tat or TNFα for 48 h. Anti-MEF2A antibody was used to immunoprecipitate the chromatin and the fold enrichment of lims1/pinch promoter relative to the matched input control was quantified by q-PCR. (I) Luciferase activity was measured in Scr siRNA and Cav3.1 KD neurons transfected with lims1/pinch luciferase construct after treatment with or without Tat or TNFα for 48 h. Data represent mean ± SEM; **P < 0.01; ***P < 0.001; n = 3–5 (one-way ANOVA).