Additional file 3 of Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

Additional file 3: Supplementary Figure S3. Cav3.1 KD blocks PINCH induction and preserves neuronal mitochondrial distribution. (A) Cellular actin architecture in Scr siRNA and Cav3.1 knockdown (KD) neurons untreated or exposed to Tat or TNFα for 48 h using confocal microscopy. Neurons were fixed, permeabilized and labeled with anti-PINCH and stained with phalloidin. Representative confocal images show preserved actin polymerization in Cav3.1 KD neurons exposed to Tat or TNFα. (B and C) Quantification of the PINCH fluorescence (B) and the actin filament length (C). (D) Quantification of relative protein abundance (phospho-Cofilin/Cofilin) and representative Western blots of lysates from Scr siRNA and Cav3.1 KD human neurons untreated or exposed to Tat or TNFα. (E) Cell lysates from Scr siRNA and Cav3.1 KD neurons untreated or exposed to Tat or TNFα were immunoprecipitated with anti-kinesin antibody. Following immunoprecipitation, total cell lysates (input; left) and immunoprecipitated materials (IP; right) were subjected to Western blot analysis. Samples were probed with antibodies against Tubulin, kinesin and Miro1. (F) Mitochondrial distribution was observed in Scr siRNA and Cav3.1 KD neurons untreated or exposed to Tat or TNFα for 48 h using confocal microscopy. Neurons were stained with dihydrorhodamine (DHR123) and changes in mitochondrial distribution were observed. Representative confocal images show preserved mitochondrial distribution in Cav3.1 KD neurons exposed to Tat or TNFα. (G) Quantification of the distance (μm) of mitochondria from the nucleus. Data represent mean ± SEM; ***P < 0.001; n = 3–5 (one-way ANOVA).