Fig 1: Mitochondria are unaffected by TDP-43 silencing in HeLa cells. (a) TOM20 expression was analysed by WB in lysates of HeLa cells silenced, or not (scramble), for TDP-43 expression. Both a representative WB (left panel) and the densitometric analysis for TOM20 expression (right panel) are reported; n = 8 for each cell type, p-value > 0.1. (b) Representative confocal images of TDP-43-silenced or control (scramble) HeLa cells stained with antibodies to TOM-20 and to TDP-43. Nuclei were visualised by Hoechst staining. Scale bar, 20 µm. (c) The box plot reports the values of mitochondrial circularity (see Materials and Methods) in both cell types. n = 41 cells for each condition, p-value > 0.06. (d,e) The relative abundance of MCU, MICU1 and MICU2 mRNAs (d) or proteins (e) were evaluated by RT-PCR or WB, respectively. In (e), representative WBs (left panel) and densitometric analyses of immunoreactive bands (right panel) are reported. n = 5, p-value > 0.5 (d); n = 4, p-value > 0.2 (e). Mann–Whitney U test for all statistics.
Fig 2: Altered mitochondrial function and MCU expression in ERMS cell lines and patient samples.A, B Basal and maximal mitochondrial Ca2+ levels in HSMM, RD, RD18, JR1, RH30 and RH41 cells was measured with Rhod2-AM staining. The graph on the right below shows basal and maximal mitochondrial Ca2+ uptake upon induction with 100 µM histamine (n = 3). Values correspond to the average ± SEM. The blue line indicates significance calculated by comparing the average of ERMS cell lines and ARMS cell lines. C, D Oxygen consumption rate (OCR) was measured with Seahorse analyser with the addition of O: Oligomycin, FCCP: Carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone, AA + R: Antimycin A and Rotenone accordingly. Values correspond to average ± SEM (n = 3). Basal and maximal respiration rate of HSMM, ERMS and ARMS cell lines as well as mitochondrial ATP-linked respiration are shown (n = 3). The blue line shows significance between the average of ERMS cell lines and ARMS cell lines. E MCU mRNA was examined in HSMM, RD, RD18, JR1, RH30 and RH41 by qPCR analysis (n = 3). Values correspond to average ± SEM. Statistical significance was calculated by one-way ANOVA analysis. The blue line shows the significance comparing the average of ERMS cell lines and ARMS cell lines. F Western blot analysis showing MCU, MICU1 and HSP60 protein levels in HSMM, RD, RD18, JR1, RH30 and RH41 cells. ß-actin was used as loading control. A representative image of three independent experiments is shown. G 6 archival ERMS patient tumour specimens were analysed by IHC using anti-MCU antibody. Images were taken at ×40 magnification. Inset shows ×3 zoomed in image. Scale bar: 50 µm. One-way ANOVA test with appropriate correction was performed for statistical analysis. ns not significant, *p = 0.05, **p = 0.01, ***p = 0.001 and ****p = 0.0001.
Fig 3: (A) Representative western blots and (B–E) quantification showing the expression levels of (B) MCU, (C) MCUR1, (D) MICU1, and (E) MICU2 in human endothelial cells under in vitro OGD conditions at different time points as indicated. ß-action was used as a loading control. mean ± SEM, n = 5, ns = non-significant; *P < 0.05; **P < 0.01.
Fig 4: MCU regulates mitochondrial functions in ERMS.A Western blot analysis showed significant downregulation of MCU expression in stable MCU knockdown RD cells with no change in MICU1, MICU2 and HSP60 expression. The western blot is representative of three independent experiments. B, C Co-localisation of Rhod2-AM and MitoTracker staining. Scales bar: 10 µm. Basal and maximal mitochondrial Ca2+ uptake upon induction with 100 µM histamine using Rhod2-AM staining is shown in control and shMCU cells (n = 3). Values correspond to the average ± SEM. D Mitochondrial hydrogen peroxide measurement using pC1-HyperRed-mito fluorescent probe. Scale bar: 5 µm. Fluorescence intensity quantified with Image J (n = 3). Values correspond to average ± SEM. E MitoSOX Red staining of shMCU in comparison to shScr is shown (n = 5). Values correspond to average ± SEM. F Cellular ROS in shMCU as compared to shScr was measured by flow cytometry using CM-H2DCFDA staining (n = 3). Values correspond to average ± SEM. G ATPlite kit revealed reduced ATP production in shMCU as compared to shScr (n = 4). Values correspond to average ± SEM. H, I OCR was measured in shMCU cells compared to shScr cells. O: Oligomycin, FCCP: Carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone, AA + R: Antimycin A and Rotenone were added accordingly. Values correspond to average ± SEM (n = 3). Basal and maximal respiration rate along with ATP-linked respiration in shScr and shMCU cells is shown. Two-tailed non-parametric unpaired t test was performed for statistical analysis. ****p = 0.0001.
Supplier Page from MilliporeSigma for Anti-MICU1 antibody produced in rabbit