Journal: The EMBO Journal

Article Title: Reticulophagy receptor FAM134C restrains BMP receptor signaling

doi: 10.1038/s44318-025-00581-3

Figure Lengend Snippet: ( A ) Amino acids withdrawal attenuates BMP-induced Id1-luc reporter activity. A549 cells were transfected with Id1-luc reporter plasmids, after 24 h change medium to complete culture medium or nutrient depleted medium (without amino acids or FBS) with or without BMP2 (50 ng/ml) for 8 h. Relative luciferase activity was determined using a microplate-type luminometer with luciferase assay reagent. Statistical analysis by unpaired two-tailed Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SD ( n = 3 independent experiments). After BMP stimulation, Medium vs -FBS p = 8.39E-03, ( B ) mTOR inhibition attenuates BMP-induced Id1-luc reporter activity. A549 cells were transfected with Id1-luc reporter plasmids, after 24 h cells stimulated with Torin1, Rapamycin or MHY1486 and together with or without BMP2 for 8 h. Relative luciferase activity were determined using a microplate-type luminometer with luciferase assay reagent. Statistical analysis by unpaired two-tailed Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SD ( n = 3 independent experiments). After BMP stimulation, DMSO vs Torin1 p = 4.34E-04; DMSO vs Rapamycin p = 1.35E-04, ( C ) Amino acids withdrawal blocks BMP signaling. A549 cells were cultured with complete culture medium or amino acids withdrawal medium for 4 h, then treated with BMP2 (50 ng/ml) for 1 or 24 h. Expression of p-Smad1/5/8, Smad1, Id1, p-mTOR, mTOR, p-S6K, S6K, BMPR1A, and GAPDH were measured by Western blotting. ( D ) mTOR inhibition blocks BMP signaling. A549 cells were stimulated with Torin1, Rapamycin or MHY1485 and together with or without BMP2 for 1 or 24 h. Expression of p-Smad1/5/8, Smad1, p-mTOR, mTOR, p-S6K, S6K, BMPR1A, and GAPDH were measured by Western blot. ( E ) FAM134C degrades BMPR1A by autophagy. HEK293T cells were co-transfected with vectors encoding FAM134C-FLAG or Smurf1-FLAG and BMPR1A-HA, 24 h after transfection, cells were treated with MG132 (20 μM), CQ (10 μM), or 3-MA (200 μM) for 4 h, and expression of FAM134C, Smurf1 and BMPR1A were measured by Western blotting. ( F ) FAM134C promotes BMPR1A degradation and inhibits Smad1/5/8 phosphorylation. U2OS cells were stably expressing FAM134C-FLAG or GFP-FLAG as a control, pretreated with CQ (10 μM) or Rapamycin (100 nM) for 4 h, and then treated with BMP2 (50 ng/ml) for 1 h. Expression of BMPR1A, p-Smad1/5/8, Smad1, FAM134C, LC3, and GAPDH were measured by Western blotting. ( G ) FAM134C knockout stabilizes BMPR1A. U2OS or U2OS FAM134C knockout cells were treated with Rapamycin (100 nM) and BMP2 (50 ng/ml) for 1 or 24 h. Expression of BMPR1A, p-Smad1/5/8, Smad1, FAM134C, LC3, and GAPDH were measured by Western blotting. ( H ) ATG5 knockdown blocks FAM134C-dependent degradation of BMPR1A. ATG5 stable knockdown U2OS cells were transfected with FAM134C-FLAG or the FLAG vector. Twenty-four hours after transfection, cells were treated with BMP2 (50 ng/ml) for 0.5 or 1 h. Expression of BMPR1A, p-Smad1/5/8, Smad1, FAM134C, and GAPDH were measured by Western blotting. ( I ) FAM134C is colocalized with BMPR1A in lysosomes. U2OS cells were co-transfected with GFP-FAM134C and mCherry-BMPR1A. After treatment with CQ (10 μM) 4 h, cells were analyzed by immunofluorescence for FAM134C and BMPR1A, and lysosomes (labeled by an anti-LAMP1 antibody) using Zeiss LSM880. DAPI (blue fluorescence) was used to stain nuclei (scale bar, 10 μm). Percentage of FAM134C colocalized with BMPR1A in the lysosome was quantified by ImageJ. Statistical analysis by unpaired two-tailed Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SD ( n = 3 independent experiments; each with five technical replicates). Vector vs FAM134C p = 1.00E-02. .

Article Snippet: Torin1 , MedChemExpress , Cat# HY-13003.

Techniques: Activity Assay, Transfection, Luciferase, Two Tailed Test, Inhibition, Cell Culture, Expressing, Western Blot, Phospho-proteomics, Stable Transfection, Control, Knock-Out, Knockdown, Plasmid Preparation, Immunofluorescence, Labeling, Fluorescence, Staining

Journal: Cell Death Discovery

Article Title: mTORC1 accelerates osteosarcoma progression via m 6 A-dependent stabilization of USP7 mRNA

doi: 10.1038/s41420-024-01893-9

Figure Lengend Snippet: A , B The protein expression levels of p-S6, S6, p-S6K, and S6K in MG63 and U2OS cells after treatment with 0–1000 nM of E2 for 48 h. C The protein expression levels of MTTLE3, METTL14, and WTAP in U2OS cells after treatment with or without 100 nM of E2 for 48 h. D The protein expression levels of MTTLE3, METTL14, and WTAP in U2OS cells after treatment with 100 nM of E2 for 48 h with or without pretreatment of 1 μM of torin1. E LC–MS analysis of m6A levels in U2OS cells treated with with 100 nM of E2 for 48 h with or without pretreatment of 1 μM of torin1. F , G Determination and quantification of ASC speck formation by immunofluorescence staining in 100 nM of E2 treated U2OS cells with or without torin1, scale bar: 50 µm. Arrows signify ASC speck. H The mRNA expression levels were determined following following shCtrl, shWTAP#1, and shWTAP#2 transfection. I The relative cell proliferation in shCtrl, shWTAP#1, and shWTAP#2 transfected U2OS cells for 0–96 h. The EdU staining ( J ) and EdU positive cells ( K ) in shCtrl, shWTAP#1, and shWTAP#2 transfected U2OS cells for 48 h, scale bar: 50 µm. L , M The migration capacity and migrated cell number in shCtrl, shWTAP#1, and shWTAP#2 transfected U2OS cells for 24 h, scale bar: 50 µm. N LC–MS analysis of m6A levels in U2OS cells treated with with 100 nM of E2 following shCtrl or shWTAP#2 transfection. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs control group, ## P < 0.01, #### P < 0.0001 vs E2 group.

Article Snippet: The cover slides were fixed by 4% paraformaldehyde after incubation with phosphate buffered saline (PBS), E2 (100 nmol/L), Torin1 (1 μM/L), E2+ Torin1 treatment for 24 h. 0.5% Triton X-100 was added for cell permeability for 20 min. After blocking, the slides or or OS tissue sections were incubated with the primary antibodies, anti-ASC speck (1:200, Santa Cruz, CA, USA) at 4 °C overnight, respectively.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Staining, Transfection, Migration, Control

Journal: Cell Death Discovery

Article Title: mTORC1 accelerates osteosarcoma progression via m 6 A-dependent stabilization of USP7 mRNA

doi: 10.1038/s41420-024-01893-9

Figure Lengend Snippet: A The SDS-PAGE band pattern of immunoprecipitated proteins using anti-USP7 and IgG. The bands of protein were excised to mass spectrometry (MS) analysis for identification. The red arrow marks the distinct band (NLRP3) in USP7 immunoprecipitated samples. B The immunoprecipitated protein of NLRP3 were analyzed using anti-USP7 and IgG. C The protein expression levels of NLRP3, pro-caspase1, and caspase1 in MG63 and U2OS cells treated with 100 nM of E2 for 48 h with or without pretreatment of 1 μM of torin1. D The protein expression levels of NLRP3 and USP7 in U2OS cells treated with shCtrl or shUSP7 for 24 h. E U2OS cells were transfected with shCtrl or shUSP7 for 24 h. The protein extracts were immunoprecipitated with IgG beads of anti-NLRP3, and the protein expression levels of K48-ubiquitin, K63-ubiquitin and NLRP3 were detected. F The relative expression of NLRP3 in OS tissues and normal tissues ( n = 8). G The correlation between USP7 and NLRP3 in OS tissues or normal tissues ( n = 8). H The colabel staining of USP7 and NLRP3 in OS tissues or normal tissues, scale bar: 50 µm. Arrow indicates USP7 + NLRP3 + cells. I Quantification of the USP7 + NLRP3 + cells in OS tissues or normal tissues ( n = 8). ** P < 0.01, **** P < 0.0001.

Article Snippet: The cover slides were fixed by 4% paraformaldehyde after incubation with phosphate buffered saline (PBS), E2 (100 nmol/L), Torin1 (1 μM/L), E2+ Torin1 treatment for 24 h. 0.5% Triton X-100 was added for cell permeability for 20 min. After blocking, the slides or or OS tissue sections were incubated with the primary antibodies, anti-ASC speck (1:200, Santa Cruz, CA, USA) at 4 °C overnight, respectively.

Techniques: SDS Page, Immunoprecipitation, Mass Spectrometry, Expressing, Transfection, Ubiquitin Proteomics, Staining

Journal: STAR Protocols

Article Title: Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) to identify chromatin-interactome in prostate cancer cells

doi: 10.1016/j.xpro.2022.101434

Figure Lengend Snippet:

Article Snippet: Torin 1 , Toronto Research Chemicals , Cat#T548700.

Techniques: Control, Recombinant, Sequencing, Modification, Purification, Software