rabbit anti mt co1  (Boster Bio)

Journal: Journal of Translational Medicine

Article Title: Targeting the MARCH5-MFN2 axis to enhance mitochondrial fusion and sensitize multiple myeloma cells to venetoclax

doi: 10.1186/s12967-025-06942-0

Figure Lengend Snippet: 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

Article Snippet: Membranes were then blocked and incubated with the following primary antibodies: MARCH5 (#PA5-25584) (Thermo Fisher Scientific, Waltham, MA, USA); MFN2 (# 9482S), FLAG(#F3165), MFF(#84580S), DRP1 (#14647S), UBIQUITIN (#3933S), PARP (#9532), CASPASE 3 (#9665P), CASPASE 8 (#9746P), BAD (#9239), BIK (#4592), BAK(#12105), BID(#2002), BIM (#2933), NOXA (#14766), NDUFS1 (#70264), NDUFAB1 (#71814), UQCRFS1 (#95231), COX1 (#55159), COX IV (#4850) were from Cell Signaling Technology (Denvers, MA, USA); PUMA (SC-374223) was from Santa Cruz Biotechnology (Dallas, TX, USA). γ-Tubulin (T6557; Sigma-Aldrich, St. Louis, MO, USA), GAPDH (#5174; Cell Signaling Technology, Denvers, MA, USA) and α-TUBULIN (#2125S; Cell Signaling Technology, Denvers, MA, USA) were used as loading controls.

Techniques: Phospho-proteomics, Generated, Quantitative RT-PCR, Expressing, Electroporation, Negative Control, Western Blot, Control, Cell Counting, Flow Cytometry, Staining

Journal: Journal of Translational Medicine

Article Title: Targeting the MARCH5-MFN2 axis to enhance mitochondrial fusion and sensitize multiple myeloma cells to venetoclax

doi: 10.1186/s12967-025-06942-0

Figure Lengend Snippet: Promotion of mitochondrial fusion via genetic MFN2 upregulation replicates MARCH5 loss and triggers anti-myeloma responses. A Western blot analysis of NDUFS1, COX1, COX4, and MFN2 proteins in independent AMO-BZB cell clones (CL.15 and CL.19) with doxycycline-inducible MFN2 expression. GAPDH was used as a loading control. B Histogram bars reporting real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO-BZB cell clones (CL.19) with doxycycline-inducible MFN2 expression, 72 h after treatment. The graph reports basal respiration, spare capacity, maximal respiration, leak state, ATP production, and non-mitochondrial respiration. * p < 0.05. C) Cell viability was assessed by Cell Titer Glo assay in AMO-BZB cell clones (CL.15 and CL.19) with doxycycline-inducible MFN2 expression, 6 and 9 days after treatment with doxycycline. * p < 0.05; ** p < 0.01. D Flow cytometry analysis of Annexin V/7AAD-stained AMO-BZB cell clone (CL.19) with doxycycline-inducible MFN2 expression, 6 and 9 days after treatment with Doxycycline. E Western blot analysis of PUMA and NOXA expression in AMO-BZB cell clones (CL.15; CL.19); GAPDH was used as loading control. F In vivo tumor growth evaluation of subcutaneous xenografts with AMO-BZB (CL.19), receiving doxycycline or vehicle as control; administrations were performed via drinking water. Average ± SD of the tumor volume for each group is shown; p -values were obtained using two-tailed t-test. ** p < 0.001. G Kaplan–Meier curves relative to AMO-BZB xenografts (CL.19) receiving doxycycline or vehicle as control (log-rank-test). Survival was evaluated from the first day of treatment until death or sacrifice. Percentage of mice alive is shown. H Western blot analysis of MFN2 protein levels in tumors retrieved from doxycycline or vehicle-treated mice; α-tubulin was used as loading control. Densitometric analysis of protein levels represents the mean ± SD from doxycycline-or vehicle-treated mice after normalization. I IHC analysis (20 × magnification) of Ki-67 expression in xenografts from AMO-BZB cells (CL.19) with doxycycline-inducible MFN2, retrieved from mice before sacrifice; representative images are reported. L Western blot analysis of BAD, BID, NOXA and PUMA protein levels in retrieved xenografts; GAPDH was used as loading control. Densitometric analysis of protein levels represents the mean ± SD from doxycycline-or vehicle-treated mice after normalization * p < 0.05; ** p < 0.01

Article Snippet: Membranes were then blocked and incubated with the following primary antibodies: MARCH5 (#PA5-25584) (Thermo Fisher Scientific, Waltham, MA, USA); MFN2 (# 9482S), FLAG(#F3165), MFF(#84580S), DRP1 (#14647S), UBIQUITIN (#3933S), PARP (#9532), CASPASE 3 (#9665P), CASPASE 8 (#9746P), BAD (#9239), BIK (#4592), BAK(#12105), BID(#2002), BIM (#2933), NOXA (#14766), NDUFS1 (#70264), NDUFAB1 (#71814), UQCRFS1 (#95231), COX1 (#55159), COX IV (#4850) were from Cell Signaling Technology (Denvers, MA, USA); PUMA (SC-374223) was from Santa Cruz Biotechnology (Dallas, TX, USA). γ-Tubulin (T6557; Sigma-Aldrich, St. Louis, MO, USA), GAPDH (#5174; Cell Signaling Technology, Denvers, MA, USA) and α-TUBULIN (#2125S; Cell Signaling Technology, Denvers, MA, USA) were used as loading controls.

Techniques: Western Blot, Clone Assay, Expressing, Control, Glo Assay, Flow Cytometry, Staining, In Vivo, Two Tailed Test