Journal: Developmental Cell

Article Title: Sestrin2 drives ER-phagy in response to protein misfolding

doi: 10.1016/j.devcel.2024.07.004

Figure Lengend Snippet: ER storage promotes TFEB nuclear translocation via the upregulation of the leucine sensor Sestrin2 (A) Venn diagram showing that 73 genes were commonly regulated in RCS and HeLa cells upon COL2A1 R789C and COL1A2 G610C expression, respectively. (B) Heatmap of the genes whose silencing significantly reverted TFEB nuclear localization in HeLa GFP-TFEB cells expressing COL1A2 G610C. A low score of nuclear TFEB localization is represented in violet, a high score in pink. Values represent the mean of N = 8 biological replicates. (C)Quantitative real-time PCR analysis of Sestrin2 ( SESN2 ) in Amish, PiZ, and kEDS primary cells. Values were normalized to CYC (Amish and PiZ) and HPRT (kEDS and PiZ) gene expression and shown as fold change relative to their corresponding controls. Mean ± standard error of mean (SEM) of N = 3 biological replicates in Amish, N = 4 biological replicates in PiZ, and N = 3 biological replicates in kEDS. Student’s unpaired t test: ∗ p < 0.05; ∗∗ p < 0.005. (D–F) Western blot analysis of (D) phospho-TFEB (S211) and phosho-P70S6K (T389) in HeLa cells and (E) phospho-ULK1 (S757) in U2OS cells expressing COL1A2 G610C. Torin 1 was used as a positive control. Actin and FILAMIN were used as loading controls. In (F), the bar graphs show quantification of the indicated proteins as fold change relative to untransfected cells. Mean ± SEM of N = 3 independent experiments. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test: ∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005; ∗∗∗∗ p < 0.0001. (G) Immunofluorescence staining of mTORC1 (green) and lysosomes (LAMP1, red) in U2OS cells expressing GFP-COL1A2 G610C. Inset, GFP-COL1A2 G610C immunostaining. Small right panels are magnifications of the boxed areas. Scale bars, 10 μm. The bar graph shows co-localization of mTORC1 with LAMP1 (expressed as Manders’ coefficient). Mean ± SEM of N = 3 independent experiments. n = 20 control and GFP-COL1A2 G610C transfected cells. Student’s unpaired t test: ∗∗ p < 0.005. (H) Subcellular localization of endogenous TFE3 (red) in WT and KICSTOR KO HEK293T cells transfected with GFP-COL1A2 G610C (green) or treated with Torin 1. Scale bars, 20 μm. The bar graph (bottom) shows quantification of cells with nuclear TFE3. Mean ± SEM of N = 3 independent experiments. n = 40 cells. One-way ANOVA with Šídák’s multiple comparisons test: ∗∗∗∗ p < 0.0001; ns ≥ 0.05. (I) Representative immunofluorescence of FLAG-TFEB (red) subcellular localization in U2OS cells expressing GFP-COL1A2 G610C cells (green) without or with leucine (1.2 mM, 1 h). The dotted circle indicates nuclei. Scale bars, 7 μm. The bar graph shows quantification of % cells with nuclear TFEB relative to control. Mean ± SEM of N = 3 independent experiments. n = 40 cells for each treatment. One-way ANOVA with Dunnett's multiple comparisons test: ∗∗∗ p < 0.0005; ns ≥ 0.05. (J) Immunofluorescence analysis of TFE3 (green) subcellular localization in PiZ hepatocytes untreated (control) or treated with leucine (4 mM, 3 h). The dotted circle indicates nuclei. Scale bars, 20 μm. Bar graph showing quantification of the percentage of cells with nuclear TFE3 (right). Mean ± SEM of N = 3 independent experiments. n = 292 control and PiZ hepatocytes. Student’s unpaired t test: ∗∗∗∗ p < 0.0001. (K) Representative fluorescence microscopy analysis of PiZ hepatocytes transfected with the ssRFP-GFP-KDEL plasmid, left untreated (control) or treated with leucine (4 mM, 3 h). Scale bars, 10 μm. Bar graph shows the ratio of red-only puncta/total cells. Mean ± SEM of N = 3 biological replicates. n = 19 control and PiZ hepatocytes. Student’s unpaired t test: ∗∗∗ p < 0.0005. See also Tables S4 and .

Article Snippet: Rabbit Monoclonal ULK1 (D9D7) Antibody , Cell Signaling , Cat #6439; RRID:AB_11178933.

Techniques: Translocation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Positive Control, Immunofluorescence, Staining, Immunostaining, Control, Transfection, Fluorescence, Microscopy, Plasmid Preparation

Journal: Developmental Cell

Article Title: Sestrin2 drives ER-phagy in response to protein misfolding

doi: 10.1016/j.devcel.2024.07.004

Figure Lengend Snippet:

Article Snippet: Rabbit Monoclonal ULK1 (D9D7) Antibody , Cell Signaling , Cat #6439; RRID:AB_11178933.

Techniques: Virus, Plasmid Preparation, Retroviral, Recombinant, Clone Assay, Transfection, Bicinchoninic Acid Protein Assay, Mutagenesis, Control, Western Blot, Stable Transfection, Expressing, Isolation, Sequencing, Software, Microscopy, Imaging, Diagnostic Assay