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: E Histogram bars represent real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO cells, 48 h after treatment with Leflunomide.

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: E Histogram bars represent real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO cells, 48 h after treatment with Leflunomide.

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

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: Induction of MFN2 via leflunomide replicates MARCH5 loss and triggers anti-MM responses. A Western blot analysis of MFN2 was performed in AMO cells, 72 h after treatment with increasing doses of Leflunomide or DMSO as vehicle; GAPDH was used as loading control. B Heatmap shows cell viability assessed by Cell Titer Glo assay in MM cells treated with Leflunomide for 48 h. Viable cells are reported as percentage of vehicle. C Cell viability assessed by Cell Titer Glo assay in AMO and H929 cells treated with Leflunomide for 72 h, and then co-cultured with HS5 cells for 48 h. Viable cells were expressed as the percentage of vehicle. Data represent the average ± SD of three independent experiments. * p < 0.05. D Western blot analysis of NDUFS1, NDUFAB1, and COX4 expression in AMO cells, 72 h after treatment with Leflunomide; GAPDH was used as loading control. E Histogram bars represent real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO cells, 48 h after treatment with Leflunomide. The graph reports basal respiration, spare capacity, maximal respiration, leak state, ATP production, and non-mitochondrial respiration. * p < 0.05. F Western blot analysis of PUMA and NOXA expression in AMO cells, 72 h after treatment with Leflunomide; GAPDH was used as loading control. G In vivo tumor growth evaluation of subcutaneous AMO xenograft receiving i.p. Leflunomide (20 mg/kg), or vehicle as control; administrations were performed 5 days a week for a total of four weeks. Average ± SD of the tumor volume for each group is shown; p -values were obtained using two-tailed t-test. * p < 0.05. H Kaplan–Meier curves relative to i.p. Leflunomide-treated AMO xenografts compared to control xenografts (log-rank-test, * p = 0.0094). Survival was evaluated from the first day of treatment until death or sacrifice. Percentage of mice alive is shown. I Western blot analysis of MFN2 protein levels in tumors retrieved from Leflunomide- or vehicle-treated mice; α-tubulin was used as loading control. Densitometric analysis of protein expression represents the mean ± SD from Leflunomide-or vehicle-treated tumor xenografts after normalization. ** p < 0.01. L IHC analysis (20 × magnification) of Ki-67 expression in AMO xenografts retrieved from mice, four weeks after Leflunomide treatment; representative images are reported. M Western blot analysis of BAK, BIK, BIM, BID, NOXA and PUMA protein levels in tumors retrieved from Leflunomide-or vehicle-treated mice; α-tubulin was used as loading control. Densitometric analysis of protein levels represents the mean ± SD from Leflunomide-or vehicle-treated mice after normalization

Article Snippet: E Histogram bars represent real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO cells, 48 h after treatment with Leflunomide.

Techniques: Western Blot, Control, Glo Assay, Cell Culture, Expressing, In Vivo, Two Tailed Test

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 influences Venetoclax response in MM cells. A Cell viability was assessed by CTG assay in AMO, AMO-BZB, H929, and H929-BZB cells 48 h after treatment with increasing doses of Venetoclax compared to vehicle (DMSO). B Cell viability was assessed by CTG assay in AMO-BZB cells after electroporation with 100 nM siMARCH5#1 or negative control (NC) siRNA, alone or in combination with Venetoclax. Viable cells are expressed as a percentage relative to vehicle-treated cells. * p < 0.05; ** p < 0.001. C Heatmap showing combination indexes (CI), determined using SynergyFinder software, 48 h after combined treatment with Venetoclax and Leflunomide in AMO cells. D Cell viability was assessed by CTG assay in AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or the corresponding empty vector (EV), 48 h after treatment with increasing doses of Venetoclax compared to vehicle (DMSO). ** p < 0.01. E Histogram showing real-time Oxygen Consumption Rate (OCR) measurements using the OROBOROS system in AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, 48 h after Venetoclax treatment. The graph reports basal respiration, spare respiratory capacity, maximal respiration, leak state, ATP production, and non-mitochondrial respiration. * p < 0.05; ** p < 0.01. F Flow cytometry analysis of mitochondrial ROS levels in AMO cells stained with MitoSOX Red after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, followed by Venetoclax treatment. Histogram is representative of three independent experiments. Mean fluorescence intensity (MFI) values are reported. * p < 0.05; ** p < 0.01. G Flow cytometry analysis of Annexin V/7-AAD-stained AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, treated alone or in combination with increasing doses of Venetoclax. H Western blot analysis of PARP1 and CASPASE 3 cleavage in AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, 48 h after treatment with Venetoclax or vehicle (DMSO). GAPDH was used as a loading control. I Flow cytometry analysis of mitochondrial membrane potential using TMRM staining, in AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, followed by Venetoclax treatment. Histogram is representative of three independent experiments. MFI values are reported. * p < 0.05; ** p < 0.01

Article Snippet: E Histogram bars represent real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO cells, 48 h after treatment with Leflunomide.

Techniques: CTG Assay, Electroporation, Negative Control, Software, Expressing, Plasmid Preparation, Flow Cytometry, Staining, Fluorescence, Western Blot, Control, Membrane

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: 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.

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: 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.

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

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: Induction of MFN2 via leflunomide replicates MARCH5 loss and triggers anti-MM responses. A Western blot analysis of MFN2 was performed in AMO cells, 72 h after treatment with increasing doses of Leflunomide or DMSO as vehicle; GAPDH was used as loading control. B Heatmap shows cell viability assessed by Cell Titer Glo assay in MM cells treated with Leflunomide for 48 h. Viable cells are reported as percentage of vehicle. C Cell viability assessed by Cell Titer Glo assay in AMO and H929 cells treated with Leflunomide for 72 h, and then co-cultured with HS5 cells for 48 h. Viable cells were expressed as the percentage of vehicle. Data represent the average ± SD of three independent experiments. * p < 0.05. D Western blot analysis of NDUFS1, NDUFAB1, and COX4 expression in AMO cells, 72 h after treatment with Leflunomide; GAPDH was used as loading control. E Histogram bars represent real time Oxygen Consumption Rate (OCR) measurement using OROBOROS on AMO cells, 48 h after treatment with Leflunomide. The graph reports basal respiration, spare capacity, maximal respiration, leak state, ATP production, and non-mitochondrial respiration. * p < 0.05. F Western blot analysis of PUMA and NOXA expression in AMO cells, 72 h after treatment with Leflunomide; GAPDH was used as loading control. G In vivo tumor growth evaluation of subcutaneous AMO xenograft receiving i.p. Leflunomide (20 mg/kg), or vehicle as control; administrations were performed 5 days a week for a total of four weeks. Average ± SD of the tumor volume for each group is shown; p -values were obtained using two-tailed t-test. * p < 0.05. H Kaplan–Meier curves relative to i.p. Leflunomide-treated AMO xenografts compared to control xenografts (log-rank-test, * p = 0.0094). Survival was evaluated from the first day of treatment until death or sacrifice. Percentage of mice alive is shown. I Western blot analysis of MFN2 protein levels in tumors retrieved from Leflunomide- or vehicle-treated mice; α-tubulin was used as loading control. Densitometric analysis of protein expression represents the mean ± SD from Leflunomide-or vehicle-treated tumor xenografts after normalization. ** p < 0.01. L IHC analysis (20 × magnification) of Ki-67 expression in AMO xenografts retrieved from mice, four weeks after Leflunomide treatment; representative images are reported. M Western blot analysis of BAK, BIK, BIM, BID, NOXA and PUMA protein levels in tumors retrieved from Leflunomide-or vehicle-treated mice; α-tubulin was used as loading control. Densitometric analysis of protein levels represents the mean ± SD from Leflunomide-or vehicle-treated mice after normalization

Article Snippet: 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.

Techniques: Western Blot, Control, Glo Assay, Cell Culture, Expressing, In Vivo, Two Tailed Test

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 influences Venetoclax response in MM cells. A Cell viability was assessed by CTG assay in AMO, AMO-BZB, H929, and H929-BZB cells 48 h after treatment with increasing doses of Venetoclax compared to vehicle (DMSO). B Cell viability was assessed by CTG assay in AMO-BZB cells after electroporation with 100 nM siMARCH5#1 or negative control (NC) siRNA, alone or in combination with Venetoclax. Viable cells are expressed as a percentage relative to vehicle-treated cells. * p < 0.05; ** p < 0.001. C Heatmap showing combination indexes (CI), determined using SynergyFinder software, 48 h after combined treatment with Venetoclax and Leflunomide in AMO cells. D Cell viability was assessed by CTG assay in AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or the corresponding empty vector (EV), 48 h after treatment with increasing doses of Venetoclax compared to vehicle (DMSO). ** p < 0.01. E Histogram showing real-time Oxygen Consumption Rate (OCR) measurements using the OROBOROS system in AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, 48 h after Venetoclax treatment. The graph reports basal respiration, spare respiratory capacity, maximal respiration, leak state, ATP production, and non-mitochondrial respiration. * p < 0.05; ** p < 0.01. F Flow cytometry analysis of mitochondrial ROS levels in AMO cells stained with MitoSOX Red after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, followed by Venetoclax treatment. Histogram is representative of three independent experiments. Mean fluorescence intensity (MFI) values are reported. * p < 0.05; ** p < 0.01. G Flow cytometry analysis of Annexin V/7-AAD-stained AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, treated alone or in combination with increasing doses of Venetoclax. H Western blot analysis of PARP1 and CASPASE 3 cleavage in AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, 48 h after treatment with Venetoclax or vehicle (DMSO). GAPDH was used as a loading control. I Flow cytometry analysis of mitochondrial membrane potential using TMRM staining, in AMO cells after lentiviral expression of MARCH5 cDNA (OE MARCH5) or EV, followed by Venetoclax treatment. Histogram is representative of three independent experiments. MFI values are reported. * p < 0.05; ** p < 0.01

Article Snippet: 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.

Techniques: CTG Assay, Electroporation, Negative Control, Software, Expressing, Plasmid Preparation, Flow Cytometry, Staining, Fluorescence, Western Blot, Control, Membrane

Journal: The Journal of Biological Chemistry

Article Title: A CRISPR-edited isoform of the AMPK kinase LKB1 improves the response to cisplatin in A549 lung cancer cells

doi: 10.1016/j.jbc.2025.108308

Figure Lengend Snippet: Super LKB1 enhances the oxidative metabolism in A549-edited cells. A , Western blotting of HK2, LDHA, and GAPDH in cWT, c2SL+, and c3SL + cells; ( B ) normalized levels of proteins by GAPDH (a.u); ( C ) Western blotting of OXPHOS and GAPDH in cWT, c2SL+, and c3SL + cells. D , normalized levels of proteins by GAPDH (a.u); ( E ) oxygen consumption rate (OCR) by OROBOROS in cWT, c2SL+, and c3SL + cells; ( F ) comparison levels of O 2 in the cell lines in the basal condition and after treatments with oligomycin and FCCP. The data are presented as mean ± SD. Statistical analysis was performed by ANOVA followed by Dunnet’s test ∗ p < 0.05, ∗∗ p < 0.01. These data are representative of two independent experiments.

Article Snippet: The oxygen consumption rate measurement was taken at basal conditions, 1 mM oligomycin, and 1 mM FCCP in OROBOROS equipment (NextGen O2k).

Techniques: Western Blot, Comparison