Fig 2: Expression analysis of MTFR2, oxidative phosphorylation markers, glycolysis markers, ROS and H₂O₂ (CAL27, UM1). (A, B) The MTFR2 expression, oxidative phosphorylation markers (Cyt-c, SDHA, Fech, Uqcrfs1, etc.) and glycolysis markers (LDHB, Glut2, etc.) quantitative analysis. (C, D) PBS, BJO and CIS were added to detect the ROS, H2O2 expression of CAL27 and UM1. (*P < 0.05, **P < 0.01 and ***P < 0.001).

Fig 3: MARCH5 regulates mitochondrial dysfunction and oxidative phosphorylation in MM cells. A Bubble chart for GO pathway enrichment analysis showing the top 10 down-regulated datasets. Bubble chart was generated in R (v4.3.2). Combined scores are shown by the circle area, while the circle color represents the range of the adjusted p-value. B Quantitative RT-PCR analysis of MARCH5, NDUFA2, NDUFS2, NDUFA8, SDHA, UQCR11 and COX5A mRNA expression levels in AMO cells, after electroporation with 100 nM siMARCH5#1 or negative control siRNA (NC). The results show the average ± SD of mRNA expression levels after normalization with β-actin and ΔΔCt calculations. *p < 0.05; **p < 0.001. C Quantitative RT-PCR analysis of MARCH5, NDUFA2, NDUFS2, NDUFA8, SDHA, UQCR11 and COX5A mRNA expression levels in AMO-BZB cells, after electroporation with 100 nM siMARCH5#1 or negative control siRNA (NC). The results show the average ± SD of mRNA expression levels after normalization with β-actin and ΔΔCt calculations. **p < 0.001 D Western blot analysis of NDUFS1, COX1, UQCRFS1, NDUFAB1 and COX4 proteins in AMO-BZB cells, 48 h after electroporation with 100 nM siMARCH5#1, siMARCH5#2 or negative control siRNA (NC); GAPDH was used as loading control. Histogram bars show the densitometric analysis expressed as fold change relative to the NC. E Histogram bars reporting real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO-BZB cells, 48 h after electroporation with 100 nM siMARCH5#1, or NC negative control siRNA. The graph reports basal respiration, spare capacity, maximal respiration, leak state, ATP production, and non-mitochondrial respiration. *p < 0.05; **p < 0.01; F Trypan blue cell count in AMO-BZB cells 48 h after electroporation with siMARCH5#1, siMARCH5#2, siMARCH5#3, or negative control (NC) siRNA. *p < 0.05; **p < 0.01. G Flow cytometry analysis of Annexin V/7-AAD-stained AMO-BZB cells, 48 h after electroporation with 200 nM siMARCH5#1, siMARCH5#2, siMARCH5#3, or negative control (NC) siRNA. Histogram bars represent the total Annexin V-positive population, including both Annexin V-positive only and Annexin V/7-AAD double-positive cells. *p < 0.05. H–I Western blot analysis of PARP, CASPASE 3, BAD, BIK, BAK, BID, PUMA, and NOXA in AMO-BZB cells, 48 h after electroporation with 100 nM siMARCH5#1, siMARCH5#2, siMARCH5#3, or negative control (NC) siRNA. GAPDH was used as a loading control