Journal: Genes

Article Title: Interplay of Impaired Cellular Bioenergetics and Autophagy in PMM2-CDG.

doi: 10.3390/genes14081585

Figure Lengend Snippet: Figure 1. The CDG severity score is inversely correlated with autophagosome marker LC3 measured by Western blot and immunocytochemistry. (A). Western blot analysis including an inverse correlation between LC3-II/BACT and CDG severity score—NPCRS was found in PMM2-deficient patients’ fibroblasts. (B). Higher NPCRS scores (more severe CDG phenotypes) are linked to lower LC3- II/BACT values. (C). Immunocytochemistry with LC3 staining was used as a confirmatory test and further analysis of PMM2-CDG cell lines with severe, moderate, and mild phenotype. (D). Quantification of immunostaining indicated the association between the CDG phenotype severity and the LC3 intensity. P1 presents with a mild CDG score (7), P2 presents with a moderate CDG score (18), and P8 presents with a severe CDG score. Patient (P8) with severe CDG phenotype showed lower levels of LC3 staining compared to a patient (P1) with a mild CDG score. DAPI nucleus staining (blue) and LC3 staining (green).

Article Snippet: Then, cells were stained with rabbit anti-LC3 antibody (Novus Biologicals; Cat# NBP2-46892) overnight at 4 ◦C and visualized via incubation with anti-Rabbit IgG Alexa FlourTM 488 (Thermo Fischer Scientific; Cat# A-21206) for 1 h at 4 ◦C.

Techniques: Marker, Western Blot, Immunocytochemistry, Staining, Immunostaining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: TMEM9 activates Rab9-dependent alternative autophagy through interaction with Beclin1

doi: 10.1007/s00018-024-05366-1

Figure Lengend Snippet: Ectopic expression of TMEM9 does not induce LC-dependent conventional autophagy. a and b HeLa cells were co-transfected with GFP-LC3 and TMEM9-flag for 24 h, treated with 20 nM bafilomycin A 1 (Baf.A1) for 6 h, and then observed under the fluorescent microscope. Scale bar, 50 μm ( a ). The numbers of LC3 dots per cell on images in ( a ) were counted in ( b ). c and d HeLa cells were co-transfected with GFP-LC3 and TMEM9-RFP for 24 h, incubated with basal medium, EBSS medium, or 10 μM TAT-Beclin1 for 6 h, and observed under confocal microscope ( c ). Scale Bar, 5 μm. Fluorescent intensities of GFP (green) and RFP (red) on the indicated area in enlarged images of ( c ) were measured with ZEISS ZEN software ( d )

Article Snippet: The primary antibodies used include anti-LC3 (Novus Biologicals, NB600-1384), anti-Beclin1 (Cell signaling, 3495S), anti-Rab9 (Cell signaling, 5118S), anti-FLAG (Sigma Aldrich, F1804), anti-HA (Abcam, ab9110), anti-Bcl-2 (Sigma Aldrich, sc-5286), anti-αTubulin (Santa cruz technology, SC-5286).

Techniques: Expressing, Transfection, Microscopy, Incubation, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: TMEM9 activates Rab9-dependent alternative autophagy through interaction with Beclin1

doi: 10.1007/s00018-024-05366-1

Figure Lengend Snippet: TMEM9 activates Rab9-dependent alternative autophagy. a HeLa cells were transfected with control or TMEM9-flag for 24 h, treated with 20 nM of bafilomycin A 1 (Baf.A1) for the indicated times and analyzed with western blotting using anti-Rab9, anti-LC3, anti-flag, and anti-αTubulin antibodies. b – d HeLa cells were co-transfected with TMEM9-flag, GFP-Rab9, and LAMP1-mCherry in an indicated manner for 24 h and observed under the confocal microscope ( b ). The numbers ( c ) and the sizes ( d ) of Rab9-positive vesicles on the images were analyzed. The data were mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± s.d. of three independent experiments. * p < 0.05. Scale Bar, 5 μm. e and f HeLa cells were co-transfected with RFP-Rab9, Beclin1-Flag, and either TMEM9-WT-GFP or TMEM9-3NQ-GFP for 27 h, incubated in control basal (Ctrl) or EBSS medium for 6 h. Cells were immunostained with and anti-Flag antibody and observed under a confocal microscope ( e ). Scale Bar, 5 μm. Fluorescent intensities of GFP (green), RFP (red), and Alexa Fluor™ Plus 405 (Blue) on the images are measured with ZEISS ZEN software ( f )

Article Snippet: The primary antibodies used include anti-LC3 (Novus Biologicals, NB600-1384), anti-Beclin1 (Cell signaling, 3495S), anti-Rab9 (Cell signaling, 5118S), anti-FLAG (Sigma Aldrich, F1804), anti-HA (Abcam, ab9110), anti-Bcl-2 (Sigma Aldrich, sc-5286), anti-αTubulin (Santa cruz technology, SC-5286).

Techniques: Transfection, Control, Western Blot, Microscopy, Incubation, Software