gfp mff  (Addgene inc)

Journal: Science advances

Article Title: SUMOylation of MFF coordinates fission complexes to promote stress-induced mitochondrial fragmentation.

doi: 10.1126/sciadv.adq6223

Figure Lengend Snippet: Fig. 2. Mitochondrial morphology analysis and DRP1 recruitment in MFF-KO MEF cells expressing MFF-WT or MFF-K151R. (A) Confocal images of WT and MFF– knockout (KO) MEF cells stained for DRP1 and mitochondria using MitoTracker. Enlargements show zoomed section of the highlighted area. Scale bars, 10 μm. (B) MEF cell lysate probed for MFF. MFF-KO cells lack all detectable isoforms of MFF. (C) Manders’ colocalization quantification of DRP1 and MitoTracker, n = 3, 84 to 98 cells were im- aged. (D to F) Mitochondrial morphology analysis of MEF WT and MFF-KO cells. (D) number of branches per network, (E) mean mitochondrial length, and (F) free-end index. Mann-Whitney test was used to determine significance, 70 to 74 cells were imaged from three independent experiments, ****P < 0.0001. (G) Confocal images of MFF-KO MEF cells expressing either GFP, GFP-MFF-WT, or MFF-K151R. Enlargements show zoomed section of the highlighted area. Scale bars, 10 μm. (H) Viral titers of GFP, GFP-MFF-WT, or K151R infection of WT MEF cells were used to determine appropriate viral amount to infect cells with. The volume in lanes 8 and 12 were used for subse- quent experiments. (I) Manders’ colocalization of DRP1 and MitoTracker [GFP, n = 2, 52 cells; and GFP-MFF (WT and K151R), n = 3, 85 to 91 cells]. (J to L) Mitochondrial morphology analysis of MFF-KO cells expressing GFP, WT-MFF, or K151R-MFF. (J) Network branching, (K) mitochondrial length, and (L) free-end index. n = 3, 73 to 95 cells, *P < 0.05 and ***P < 0.0005, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. n.s., not significant.

Article Snippet: 10, eadq6223 (2024) 4 October 2024 12 of 15 Lentiviral GFP- MFF constructs were produced in the plasmid pXLG3- PX- GFP- WPRE. pAcGFP- tagged human MFF isoform 1 was subcloned from pAcGFP- C1- MFF [a gift from G. Voeltz (Addgene, plasmid no. 49153)] by digestion of pAcGFP- C1- MFF with Nhe I and Bam HI to isolate the pAcGFP- MFF insert and ligating into Spe I and Bam HI cut pXLG3- PX- GFP- WPRE in place of the GFP.

Techniques: Expressing, Knock-Out, Staining, MANN-WHITNEY, Infection

Journal: Science advances

Article Title: SUMOylation of MFF coordinates fission complexes to promote stress-induced mitochondrial fragmentation.

doi: 10.1126/sciadv.adq6223

Figure Lengend Snippet: Fig. 5. AMPK activation enhances MFF SUMOylation and MiD51 displacement in response to CCCP. (A) Confocal images of HEK293T cells transfected with mito- DsRed following 1 hour of treatment with 10 μM CCCP. Scale bars, 10 μm. (B) HEK293T cells were transfected with CFP-MFF (WT or K151R) and treated with 10 μM CCCP for 1 hour before lysis, either alone or in combination with the AMPK inhibitor compound C (10 μM). CFP-MFF immunoprecipitates were blotted for SUMO2/3, Ser155 phosphorylation (low- and higher-exposure blots are shown), and CFP. (C) Quantification of SUMOylation of WT-MFF following 1 hour of CCCP in the presence or absence of compound C. Representative of three independent experiments. One-sample t test for conditions versus vehicle control (Ctl), and two-sample t test for CCCP versus CCCP + CC conditions. **P < 0.01. (D and E) HEK293T cells were transfected with CFP-MFF (WT) and treated with rotenone (250 ng/ml) or 1 mM AICAR for 1 hour before lysis. CFP immunoprecipitates were blotted for SUMO2/3 and CFP. Quantification presented in (E), n = 5 (rotenone) and n = 4 (AICAR). Uncropped blot is shown in fig. S4C. (F) HEK293T cells expressing GFP-MFF (WT or K151R) and MiD51-HA were treated with CCCP (10 μM, 1 hour). Co-immunoprecipitates were immunoblotted for HA and GFP. (G) Quantification of MiD51-HA binding to MFF following CCCP treatment, n = 4 or 5. One-sample t test for CCCP conditions versus vehicle controls, and two-sample t test for WT versus K151R CCCP conditions. *P < 0.05 and **P < 0.01.

Article Snippet: 10, eadq6223 (2024) 4 October 2024 12 of 15 Lentiviral GFP- MFF constructs were produced in the plasmid pXLG3- PX- GFP- WPRE. pAcGFP- tagged human MFF isoform 1 was subcloned from pAcGFP- C1- MFF [a gift from G. Voeltz (Addgene, plasmid no. 49153)] by digestion of pAcGFP- C1- MFF with Nhe I and Bam HI to isolate the pAcGFP- MFF insert and ligating into Spe I and Bam HI cut pXLG3- PX- GFP- WPRE in place of the GFP.

Techniques: Activation Assay, Transfection, Lysis, Phospho-proteomics, Control, Expressing, Binding Assay

Journal: Science advances

Article Title: SUMOylation of MFF coordinates fission complexes to promote stress-induced mitochondrial fragmentation.

doi: 10.1126/sciadv.adq6223

Figure Lengend Snippet: Fig. 6. MFF SUMOylation is not necessary for DRP1 recruitment under CCCP treatment but is required for promoting mitochondrial fragmentation. (A) Confocal imaging of CCCP-induced mitochondrial fragmentation in MFF-KO MEF cells virally expressing GFP alone, or WT or K151R GFP-MFF. Cells were treated with CCCP (10 μM, 1 hour). Mitochondria were stained using MitoTracker Deep Red, endogenous DRP1 stain is shown in green, and GFP channel is shown in cyan. Processed images of mi- tochondrial stain with enlargements of highlighted area. Scale bar 10 μm. (B) Manders’ colocalization analysis of DRP1 with MitoTracker. Kruskal-Wallis test, 57 to 119 cells were imaged from three independent experiments, **P < 0.01 and ****P < 0.0001. (C) Quantification of the free-end index, data were generated from three independent experiments, expressed as percentage of DMSO control, 72 to 110 cells were imaged (for GFP-MFF–expressing cells); two independent experiments, 50 to 53 cells were imaged for GFP-expressing cells. Unpaired t test, **P < 0.01.

Article Snippet: 10, eadq6223 (2024) 4 October 2024 12 of 15 Lentiviral GFP- MFF constructs were produced in the plasmid pXLG3- PX- GFP- WPRE. pAcGFP- tagged human MFF isoform 1 was subcloned from pAcGFP- C1- MFF [a gift from G. Voeltz (Addgene, plasmid no. 49153)] by digestion of pAcGFP- C1- MFF with Nhe I and Bam HI to isolate the pAcGFP- MFF insert and ligating into Spe I and Bam HI cut pXLG3- PX- GFP- WPRE in place of the GFP.

Techniques: Imaging, Expressing, Staining, Generated, Control