MARCH5 drives mitochondrial elongation... | American Society of Hematology

$MARCH5 drives mitochondrial elongation during cell-cycle progression in AML cells. (A) Primary cells from patients with AML were serially transplanted and amplified in NSG-immunodeficient mice before studying mitochondrial morphology based on their quiescence or cycling status. (B) Confocal imaging of PDXTUH93 cells using MitoTracker Deep Red (MTDR; in red), Ki-67 (in green), and DAPI (in blue) staining (scale bar, 2 μm). Quantifications of mitochondrial length in quiescent and cycling cells (right; n = 30 cells per condition). (C) PDX-AML cells were sorted using Pyronin Y and Hoechst staining. (D) Quantitative reverse transcriptase polymerase chain reaction (PCR) analysis of a targeted panel of genes involved in mitochondrial dynamics in PDX cells (n = 4 different PDX models). (E) Western blots performed on quiescent (Q) and cycling (C) fractions using anti-MFN1, -MFN2, –optic atrophy 1 (OPA1), –mitochondrial fission factor (MFF), –dynamin-like 1(DRP1), -MARCH5, and –β-actin antibodies. (F-G) Expression plasmids for wild-type MARCH-5 (MARCH5-WT) or MARCH5-H43W proteins were lentivirally transduced into OCI-AML2 cells. (F) Immunoprecipitation (IP) using the hemagglutinin (HA) tag of these proteins was performed, followed by liquid chromatography–electrospray ionization–tandem mass spectrometry quantitative proteomic analysis. Results are shown as the number of peptides for each identified protein in MARCH5-WT relative to MARCH5-H43W conditions. (G) Western blots on empty vector, MARCH5-WT, and MARCH5-H43W IP and corresponding total protein extracts (input) using anti-MFN2 and anti-HA antibodies. (H-K) MOLM-14 and OCI-AML2 cells were transduced with doxycycline (Dox)–inducible control (CLT) or anti-MARCH5 shRNAs and incubated with 1 μg/mL Dox for 4 days. (H) Electron microscopy images at 7100× original magnification. (I-K) Quantification of mitochondrial length (I), area (J), and number (K) (n = 30 cells; 5-50 mitochondria were measured in each cell). (L-O) PDX-AML cells were transduced with a vector expressing CTL or anti-MARCH5 shRNAs or a vector for MARCH5 overexpression (OE) or its empty counterpart. Confocal imaging was performed using MTDR and DAPI staining (scale bar, 2 μm). Quantifications are shown in the right panels (n = 30 cells per condition). (L) Quantitative reverse transcription PCR analysis of MARCH5 expression in PDX-AML cells transduced with CTL or anti-MARCH5 shRNAs (n = 9 different PDXs). (M) MARCH5 knockdown assays. (N) PDX-AML samples were transduced with either MARCH5 OE (M5 OE) or empty vectors. Western blot for MARCH5 and β-actin are provided. (O) M5 OE assays. Mt, mitochondria; shCTL, sh control; shM5, sh MARCH5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.$

MARCH5 drives mitochondrial elongation during cell-cycle progression in AML cells. (A) Primary cells from patients with AML were serially transplanted and amplified in NSG-immunodeficient mice before studying mitochondrial morphology based on their quiescence or cycling status. (B) Confocal imaging of PDX^TUH93 cells using MitoTracker Deep Red (MTDR; in red), Ki-67 (in green), and DAPI (in blue) staining (scale bar, 2 μm). Quantifications of mitochondrial length in quiescent and cycling cells (right; n = 30 cells per condition). (C) PDX-AML cells were sorted using Pyronin Y and Hoechst staining. (D) Quantitative reverse transcriptase polymerase chain reaction (PCR) analysis of a targeted panel of genes involved in mitochondrial dynamics in PDX cells (n = 4 different PDX models). (E) Western blots performed on quiescent (Q) and cycling (C) fractions using anti-MFN1, -MFN2, –optic atrophy 1 (OPA1), –mitochondrial fission factor (MFF), –dynamin-like 1(DRP1), -MARCH5, and –β-actin antibodies. (F-G) Expression plasmids for wild-type MARCH-5 (MARCH5-WT) or MARCH5-H43W proteins were lentivirally transduced into OCI-AML2 cells. (F) Immunoprecipitation (IP) using the hemagglutinin (HA) tag of these proteins was performed, followed by liquid chromatography–electrospray ionization–tandem mass spectrometry quantitative proteomic analysis. Results are shown as the number of peptides for each identified protein in MARCH5-WT relative to MARCH5-H43W conditions. (G) Western blots on empty vector, MARCH5-WT, and MARCH5-H43W IP and corresponding total protein extracts (input) using anti-MFN2 and anti-HA antibodies. (H-K) MOLM-14 and OCI-AML2 cells were transduced with doxycycline (Dox)–inducible control (CLT) or anti-MARCH5 shRNAs and incubated with 1 μg/mL Dox for 4 days. (H) Electron microscopy images at 7100× original magnification. (I-K) Quantification of mitochondrial length (I), area (J), and number (K) (n = 30 cells; 5-50 mitochondria were measured in each cell). (L-O) PDX-AML cells were transduced with a vector expressing CTL or anti-MARCH5 shRNAs or a vector for MARCH5 overexpression (OE) or its empty counterpart. Confocal imaging was performed using MTDR and DAPI staining (scale bar, 2 μm). Quantifications are shown in the right panels (n = 30 cells per condition). (L) Quantitative reverse transcription PCR analysis of MARCH5 expression in PDX-AML cells transduced with CTL or anti-MARCH5 shRNAs (n = 9 different PDXs). (M) MARCH5 knockdown assays. (N) PDX-AML samples were transduced with either MARCH5 OE (M5 OE) or empty vectors. Western blot for MARCH5 and β-actin are provided. (O) M5 OE assays. Mt, mitochondria; shCTL, sh control; shM5, sh MARCH5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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