egfp rab7  (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: eLife

Article Title: Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function

doi: 10.7554/eLife.100928

Figure Lengend Snippet: ( A, B ) High content microscopy (HCM) imaging and quantification of LC3 response (puncta/cell of endogenous LC3 immunofluorescent profiles) in HeLa WT , and HeLa VPS35-KO cells in response to lysosomal damage by LLOMe (1 mM, 30 and 60 min). Scale bar, 20 μm. Data, means ± SE ( n = 5), one-way ANOVA with Tukey’s multiple comparisons. ( C–E ) HCM quantification of GLUT1 response (puncta/cell of endogenous GLUT1) ( D ) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining) ( E ) in Huh7 WT treated with or without Monensin (100 µM), LLOMe (100 µM), and Bafilomycin A1 (100 nM) for 45 min. Scale bar, 20 μm. Data, means ± SE ( n = 5), one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell ( F, G ) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining), ( H ) phenotype monitored by HCM quantification in Huh7 WT cells transfected with GFP (control) or GFP-Rab7 WT , GFP-Rab7 Q67L , and GFP-Rab7 T22N , expressing plasmids. Scale bar, 20 μm. Data, means ± SE ( n = 3); one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell ( I, J ) by HCM quantification in Huh7 WT and ATG16L1-KO cells complemented with Flag (control), Flag-ATG16L1 FL , or Flag-ATG16L1 E230 , expressing plasmids. Scale bar, 20 μm. Data, means ± SE ( n = 5); one-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line/condition. Figure 7—source data 1. Numerical values for quantification in graphs.

Article Snippet: Addgene clones were: eGFP-Rab7 WT (Addgene, #12605), eGFP-Rab7 Q67L (Addgene, #28049), and eGFP-Rab7 T22N (Addgene, #28049).

Techniques: Microscopy, Imaging, Immunostaining, Transfection, Control, Expressing

Journal: eLife

Article Title: Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function

doi: 10.7554/eLife.100928

Figure Lengend Snippet: ( A, B ) High content microscopy (HCM) imaging and quantification of LC3 response (puncta/cell of endogenous LC3 immunofluorescent profiles) in HeLa WT , and HeLa VPS35-KO cells in response to lysosomal damage by LLOMe (1 mM, 30 and 60 min). Scale bar, 20 μm. Data, means ± SE ( n = 5), one-way ANOVA with Tukey’s multiple comparisons. ( C–E ) HCM quantification of GLUT1 response (puncta/cell of endogenous GLUT1) ( D ) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining) ( E ) in Huh7 WT treated with or without Monensin (100 µM), LLOMe (100 µM), and Bafilomycin A1 (100 nM) for 45 min. Scale bar, 20 μm. Data, means ± SE ( n = 5), one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell ( F, G ) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining), ( H ) phenotype monitored by HCM quantification in Huh7 WT cells transfected with GFP (control) or GFP-Rab7 WT , GFP-Rab7 Q67L , and GFP-Rab7 T22N , expressing plasmids. Scale bar, 20 μm. Data, means ± SE ( n = 3); one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell ( I, J ) by HCM quantification in Huh7 WT and ATG16L1-KO cells complemented with Flag (control), Flag-ATG16L1 FL , or Flag-ATG16L1 E230 , expressing plasmids. Scale bar, 20 μm. Data, means ± SE ( n = 5); one-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line/condition. Figure 7—source data 1. Numerical values for quantification in graphs.

Article Snippet: Addgene clones were: eGFP-Rab7 WT (Addgene, #12605), eGFP-Rab7 Q67L (Addgene, #28049), and eGFP-Rab7 T22N (Addgene, #28049).

Techniques: Microscopy, Imaging, Immunostaining, Transfection, Control, Expressing