gfp rab7 q67l  (Addgene inc)

Journal: Nature Communications

Article Title: Endosomal trafficking participates in lipid droplet catabolism to maintain lipid homeostasis

doi: 10.1038/s41467-025-57038-8

Figure Lengend Snippet: A The knockdown efficiency of Rab7a in A549 cells was assessed by western blot analysis ( p < 0.0001). B Control or Rab7a-knockdown A549 cells were fixed by 4% PFA, and then stained by Bodipy 493/503 (1 μM) and DAPI (1 μg/mL) for 30 min. The number and size of LDs, and fluorescence intensity of Bodipy 493/503 were quantified by ImageJ ( n = 30 for all figures; p < 0.0001 for number of LDs and relative Bodipy fluorescence, p = 0.3763 for average size of LDs). C Triglyceride level in control or Rab7a-knockdown A549 cells was determined ( p < 0.0001). D A549 cells were transiently transfected with GFP-Rab7a, GFP-Rab7a-T22N, or GFP-Rab7a-Q67L, and then stained with Nile red (1 μM) for 30 min. The number and size of LDs, and fluorescence intensity of Nile Red were quantified by ImageJ ( n = 13 for all figures; for number of LDs, p = 0.0097 for Rab7a T22N, p < 0.0001 for Rab7a Q67L; for average size of LDs, p = 0.4779 for Rab7a T22N, p < 0.0001 for Rab7a Q67L; and for relative Bodipy fluorescence, p < 0.0001 for both Rab7a T22N and Rab7a Q67L). Images were captured by a Zeiss 880 microscope with a 63x objective lens. The scale bar is 5 μm. The graphs represented data from three independent experiments. The statistical significance of differences was determined by using the unpaired two-tailed student’s t test, and data quantifications were expressed as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001; ns: no significance.

Article Snippet: pEGFP-C1-ADRP (addgene, #87161), mcherry-ACSL3 (addgene, #87158), GFP-Rab5 (addgene, #174454), mcherry-Rab5 (addgene, #55126), mcherry-Rab5 S23N, GFP-Rab7 (addgene, #61803), mcherry-Rab7 (addgene, #55127), GFP-Rab7 T22N (addgene, #28048), GFP-Rab7 Q67L (addgene, #28049), pCDNA3.1-Twinstrep Rab5 (Elife.

Techniques: Knockdown, Western Blot, Control, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test

Journal: Molecular and Cellular Biology

Article Title: Systematic Analysis of Human Cells Lacking ATG8 Proteins Uncovers Roles for GABARAPs and the CCZ1/MON1 Regulator C18orf8/RMC1 in Macroautophagic and Selective Autophagic Flux

doi: 10.1128/MCB.00392-17

Figure Lengend Snippet: C18orf8/RMC1 is required for endolysosomal size control and autophagic flux. (A) HeLa cells stably expressing RMC1-FLAG-HA were grown on glass coverslips, fixed, and immunostained as indicated. (B) HeLa cells were transfected with control or RMC1 siRNA pools; at 72 h posttransfection, cells were harvested for immunoblot analysis with the indicated antibodies. (C) Cells treated with siRNAs as for panel B were grown on glass coverslips, fixed, and immunostained with the late endosomal marker anti-CD63; maximum-intensity projections of z-stacks are shown, and scale bars represent 20 μm. (D) Cells treated with siRNAs as for panel B were fixed and analyzed by TEM. (E) HeLa cells were treated with individual or combined siRNAs as indicated for 72 h, followed by immunoblot analysis to evaluate depletion of RMC1 and accumulation of p62 protein. (F) Quantification of p62 levels shown in panel E for two biological replicate experiments. Error bars represent standard deviation, and significance was determined by one-way ANOVA with post hoc testing (Dunnett's multiple-comparison test). *, P < 0.05; **, P < 0.01. (G) HeLa cells were grown on glass coverslips, fixed, and immunostained with anti-CD63 and LC3B; representative maximum-intensity projections are shown, and scale bars represent 20 μm. (H) Quantification of basal LC3B punctum number in cells stained as for panel G. Data represent two pooled biological experiments, and error bars represent standard deviation. (I and J) HeLa cells treated with control or RMC1 siRNAs (as for panel E) were treated with Cell Light RFP-GFP-LC3B for 16 h, followed by incubation in nutrient-replete DMEM (I) or starvation medium (HBSS) (J) for 1 h. Autophagic flux was monitored by confocal microscopy; single z-sections are shown for each channel, and scale bars represent 20 μm. (K and L) Quantification of average percentage of RFP-GFP-positive and RFP-only puncta per cell as shown in panels I and J. Data represent two pooled biological experiments, error bars represent standard deviation of the mean, and significance was determined by one-way ANOVA with post hoc testing (Dunnett's multiple-comparison test). ****, P < 0.0001. (M) Left panel, HeLa cells were grown on glass coverslips in nutrient-replete medium, fixed, and stained with endogenous antibodies against RAB7 and CD63. Individual representative z-sections for each channel are shown, and scale bars represent 20 μm. Right panel, intensity profiles of a 20-μm line segment drawn across the z-section shown for each channel. Overlap of 488 (RAB7) and 561 (CD63) fluorescence intensity profiles (arbitrary units [AU]) indicates colocalization of RAB7 with the late endosomal marker CD63.

Article Snippet: GFP-RAB7 WT , GFP-RAB7 Q67L , and GFP-RAB7 T22N (dominant negative) were gifts from Qing Zhong (Addgene plasmids 28047, 28049, and 28048, respectively) ( 62 ).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Marker, Standard Deviation, Staining, Incubation, Confocal Microscopy, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Systematic Analysis of Human Cells Lacking ATG8 Proteins Uncovers Roles for GABARAPs and the CCZ1/MON1 Regulator C18orf8/RMC1 in Macroautophagic and Selective Autophagic Flux

doi: 10.1128/MCB.00392-17

Figure Lengend Snippet: Identification of C18orf8/RMC1 as a member of the CCZ1-MON1 GEF complex for RAB7. (A) SILAC mass spectra of heavy (control) and light (ΔRAP-enriched) C18orf8/RMC1 peptides identified in autophagosomal proteomics; K represents the heavy-labeled lysine residue. (B) Top panel, predicted domain structure of RMC1. Bottom panel, Map of high-confidence interacting proteins (HCIPs) identified in reciprocal affinity purification-mass spectrometry (AP-MS) experiments with RMC1-FLAG-HA, FLAG-HA-CCZ1, or MON1A-MYC-FLAG bait proteins expressed in 293T cells. The line thickness correlates with the number of peptides observed in each interaction. (C) GFP-CCZ1 associates with RMC1-FLAG-HA. 293T cells stably expressing RMC1-FLAG-HA were transiently transfected with FLAG-HA-GFP control or GFP-CCZ1, followed by GFP-TRAP affinity purification. Note that RMC1-FLAG-HA associates with GFP-CCZ1 but not the FLAG-HA-GFP control. (D and E) Size exclusion chromatography and AP-MS of RMC1-containing complexes. 293T extracts stably expressing RMC1-FLAG-HA were separated by size exclusion chromatography; estimated molecular mass (MM) standards corresponding to fraction number are shown. Fractions containing RMC1 and RAB7 signals were subjected to FLAG AP-MS to identify complex members. (F) GFP-RAB7 associates with endogenous RMC1. 293T cells were transiently transfected with FLAG-HA-GFP control, GFP-RAB7WT, GFP-RAB7Q67L (constitutively active), or GFP-RAB7T22N (dominant negative), followed by GFP-TRAP affinity purification and immunoblot analysis as indicated.

Article Snippet: GFP-RAB7 WT , GFP-RAB7 Q67L , and GFP-RAB7 T22N (dominant negative) were gifts from Qing Zhong (Addgene plasmids 28047, 28049, and 28048, respectively) ( 62 ).

Techniques: Labeling, Affinity Purification, Mass Spectrometry, Stable Transfection, Expressing, Transfection, Size-exclusion Chromatography, Dominant Negative Mutation, Western Blot