Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a , b Quantification of fission (Fis.) and fusion (Fus.) events associated with ER. Cells were transfected with mCherry-ER and mito-GFP. Each point represents an individual cell, with 15 cells quantified in 4 independent experiments. a ER-Positive events (%), ( b ) Total events per cell. Bars show the average ± SD. c Representative fusion event showing the enrichment of AC-ER at the fusion site (arrowhead). Cells were transfected with mCherry-mito (mitochondria, magenta) and AC-ER (actin, green). Scale bar 2 µm. d , e Quantification of fission (Fis.) and fusion (Fus.) events positive for AC-mito. Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. d ER-Positive events (%), ( e ) Total events per cell. f AC-ER signal enrichment at fusion (Left) and fission (Right) sites in cells transfected as in ( c ). Signal intensity at the event site relative to an adjacent site on the ER network was quantified for AC-ER (AC) and mCherry-ER (mCh). Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. Bars show the average ± SD. two-sided t-test. g Quantification of AC-ER signal associated with each of the fusing mitochondria (the stationary mitochondrion (Rec) and the mobile mitochondrion (Fus)) as well as with the fusion site one frame post-fusion (Post). Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. Bars show the average ± SD. One-way ANOVA. h , i Kinetics of recruitment of AC-mito and AC-ER at the fusion site showing AC-ER recruitment relative to AC-mito ( g ) and recruitment of either marker relative to the time of fusion ( h ). Each point represents an individual event (20 events total in 4 cells within 3 independent experiments). two-sided t-test.

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Marker

Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a Representative images showing the loss of AC-mito signal in primary fibroblasts transfected with AC-mito (green) and mCherry-Fis1 (magenta), and treated with the Arp2 inhibitor CK-666. Scale bar 10 µm ( b ) Quantification of AC-mito signal accumulation in cells transfected as in ( a ). Each point represents an individual cell, with 15 cells quantified per condition in 3 independent experiments. Bars show the average ± SD. Two-sided t-test. ( c ) Quantification of the number of fusion (Left, blue) and fission (Right, orange) events, as well as the fusion/fission ratio ( d ) in cells transfected as in ( a ) and treated as indicated. Each point represents an individual cell, with 12 (control) and 9 (CK-666) cells quantified in 3 independent experiments. Bars show the average ± SD. Two-sided t-test. e Quantification of fusion events as tip-to-side (Side) or tip-to-tip (End). The total number of events for 9 cells in 3 experiments is shown for each condition. f Quantification of mitochondrial length (Left) and connectivity (Right) in cells from ( c ). Each point represents an individual cell. Bars show the average ± SD. One-way ANOVA. g – i Mitochondrial fusion assay. Human primary fibroblasts were transfected with photoactivatable-GFP (PA-GFP), treated as indicated and imaged before (pre) and after activation with the 405 nm laser. Fluorescence traces ( g , Left) and the quantification of the loss of fluorescence at 3 min relative to the initial time post-activation ( g , Right) are shown. Each point ( g , Right) represents an individual cell, with 14 ctrl and 16 CK-666 cells quantified in 4 independent experiments. Bars show the average ± SD. Two-sided t-test. Fusion was also directly quantified by measuring the events in which PA-GFP was transferred from one mitochondrion to another upon fusion (H, separation by fusion type; I, total fusion events). Each point represents an individual cell, with 12 ctrl and 11 CK-666 cells quantified in 3 independent experiments. Bars show the average ± SD. Two-sided t-test.

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Control, Single Vesicle Fusion Assay, Activation Assay, Fluorescence

Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a – d Temporal relationship between mitochondrial actin and the fusion dynamin MFN2. Cells were transfected with mCherry-MFN2 and AC-mito while mitochondria were visualised with Mitotracker Deep red. ( a ) Presence of AC-mito and mCherry-MFN2 at sites of mitochondrial fission, tip-to-side fusion and tip-to-tip fusion. b – d Kinetics of recruitment of AC-mito and mCherry-MFN2 at fission ( b ) and fusion ( c ) sites showing recruitment of either marker relative to the time of fusion, as well as mCherry-MFN2 recruitment relative to AC-mito ( d ). Each point represents an individual event in 4 cells within 3 independent experiments). Bars show the average ± SD. two-sided t-test. e – h Temporal relationship between the fusion dynamin MFN2 and the fission Dynamin DRP1. Cells were transfected with mCherry-MFN2 and GFP-DRP1 while mitochondria were visualised with Mitotracker Deep red. e Presence of GFP-DRP1 and mCherry-MFN2 at sites of mitochondrial fission, tip-to-side fusion and tip-to-tip fusion. ( f – h ) Kinetics of recruitment of mCherry-MFN2 andGFP-DRP1 at fission ( f ) and fusion ( g ) sites showing recruitment of either marker relative to the time of fusion, as well as GFP-DRP1 recruitment relative to mCherry-MFN2 ( d ). Each point represents an individual event in 3 cells within 3 independent experiments). Bars show the average ± SD. two-sided t-test. ( i ) Schematic representation of the timing of MFN2 and DRP1 recruitment to tip-to-side fusion (Top) and fission (Bottom) relative to actin recruitment (AC-mito-positive).

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Marker

Journal: iScience

Article Title: Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis

doi: 10.1016/j.isci.2023.107626

Figure Lengend Snippet:

Article Snippet: Puromycin dihydrochloride , Santa Cruz Biotechnology , sc-108071.

Techniques: Staining, Virus, Plasmid Preparation, shRNA, Recombinant, Expressing, Transfection, Magnetic Beads, Protein Extraction, Bicinchoninic Acid Protein Assay, Purification, Enzyme-linked Immunosorbent Assay, Software

Journal: Advanced Science

Article Title: The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells

doi: 10.1002/advs.202512652

Figure Lengend Snippet: ITCH stabilizes APC11 by adding K27, K29, and K33‐linked polyubiquitin chains to APC11 at Lys83 in a CUL5‐dependent manner. a) Immunoblot of APC11 and ITCH in Hep3B and PLC/PRF/5 cells upon ITCH knockdown by two distinct siRNA oligos. b) Immunoblot of APC11 and APC/C substrate securin in HEK293 cells after transfection with wild‐type ITCH or its C830S mutant for 48 h. c) The stability of APC11 in Hep3B and PLC/PRF/5 cells upon ITCH knockdown. Hep3B and PLC/PRF/5 cells were transfected with indicated siRNA oligos for 72 h and then treated with CHX (100 µg mL −1 ) for the indicated time periods, followed by IB analysis. Densitometry quantifications were performed with ImageJ, and the decay curves are shown (right). d) K27/K29/K33‐linked ubiquitylation of APC11 promoted by overexpression of ITCH but not its C830S mutant. HEK293 cells were co‐transfected with indicated plasmids, followed by purification with Ni‐NTA. Pull‐downs (top) and WCE (bottom) were subjected to IB with indicated Abs. e) Co‐IP of exogenous FLAG‐tagged APC11 with endogenous ITCH or CUL5 in HEK293 cells. f) The interaction of exogenous FLAG‐tagged CUL5 with endogenous ITCH or APC11 in HEK293 cells. g) Co‐IP of endogenous ITCH with CUL5 or APC11 in Hep3B and PLC/PRF/5 cells. h) Co‐IP of exogenous FLAG‐tagged APC11 in HeLa cells upon CUL5 knockout mediated by CRISPR‐Cas9. HeLa cells with or without CUL5 were infected with lentivirus expressing FLAG‐APC11 for 72 h, and then harvested for IP analysis. Band intensities were quantified using ImageJ, and the relative ITCH‐APC11 binding affinity in sgCUL5 cells was compared to sgCtrl cells. i) Immunoblot of APC11 in sgCtrl or sgCUL5 HeLa cells after transfection with GFP‐ITCH for 48 h. Band intensities were quantified using ImageJ and expressed as relative gray values (normalized to sgCtrl/Vector cells), shown beneath each band. j,k) ITCH facilitates K27/K29/K33‐linked ubiquitylation of wild‐type APC11 but not its W14A (j) or K83R (k) mutant. HEK293 cells were co‐transfected with indicated plasmids, followed by purification with Ni‐NTA. Pull‐downs (top) and WCE (bottom) were subjected to IB with indicated Abs. Data are presented as mean ± SEM, n = 3 (c, h, i; right). For statistical analysis, significances were determined by Student's t ‐test.* p < 0.05, ** p < 0.01, ns, not significant. l A model illustrating that CUL5 stabilizes APC11 through ITCH‐mediated K27/K29/K33‐linked polyubiquitylation at Lys83.

Article Snippet: HeLa cells with CUL5 knockout by CRISPR‐Cas9 were established by puromycin (Selleck, S7417) selection after transfection with a sequence‐verified CRISPR plasmid.

Techniques: Western Blot, Knockdown, Transfection, Mutagenesis, Over Expression, Purification, Co-Immunoprecipitation Assay, Knock-Out, CRISPR, Infection, Expressing, Binding Assay, Plasmid Preparation

Journal: STAR Protocols

Article Title: Protocol for inducible piggyBac transposon system for efficient gene overexpression in human pluripotent stem cells

doi: 10.1016/j.xpro.2022.101296

Figure Lengend Snippet:

Article Snippet: Puromycin , MCE , CatHY-B1743A.

Techniques: Virus, Recombinant, Plasmid Preparation, Gel Extraction, Transfection, Software, Bacteria