Journal: Journal of Extracellular Vesicles

Article Title: Accessory ESCRT‐III proteins are conserved and selective regulators of Rab11a‐exosome formation

doi: 10.1002/jev2.12311

Figure Lengend Snippet: Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells . (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct. Proteins were detected from gels with sample loading normalised to total cell lysate protein levels and the fold change in CHMP5 knockdown cells determined. Note the more variable effects of knockdown on some of the sEV markers, but not Rab11a, in the bar chart. This is presumably the result of different levels of transient CHMP5 knockdown. Data derived from three independent experiments. (B) Nanosight tracking analysis of sEVs for diluted samples (normalised to cell lysate protein levels) shows no change in particle size and number. (C) Western blot analysis of putative exosome proteins from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct shows selective reduction in CHMP5 protein levels. The activity of mTORC1 was assessed via phosphorylation of S6 and 4E‐BP1, using phospho‐specific antibodies and a pan 4E‐BP1 antibody. Bar chart shows protein levels normalised to tubulin. Data derived from three independent experiments. (D) Western analysis of sEV preparations isolated by SEC from HCT116 colorectal cancer cells, which contain a stable IPTG‐inducible CHMP5 shRNA knockdown construct (clone #28; shCHMP5 #28), cultured in glutamine‐depleted (0.15 mM) conditions for 24 h. sEVs were collected from cells cultured in the absence (‐) or presence (+) of IPTG, both for 96 h previously and during the collection period. Putative exosome proteins were detected from gels with sample loading normalised to total cell lysate protein levels. Bar charts represent changes in levels of these putative exosome proteins relative to levels in the non‐IPTG‐induced sample. Data derived from five independent experiments. (E) Nanosight Tracking Analysis of sEVs for the shCHMP5 #28 samples produced in (D) show no change in particle size and number. (F) Western blot analysis of putative exosome proteins in EV‐secreting cells carrying a stable IPTG‐inducible CHMP5 (shCHMP5 #28) shRNA construct, under glutamine‐depleted (0.15 mM) conditions, in the presence or absence of IPTG, reveals selective reduction in CHMP5 protein levels. Bar charts derived from three independent experiments. All data for bar charts were analysed by the Kruskal‐Wallis test: * p < 0.05; n.s. = not significant. Bars and error bars denote mean ± SD

Article Snippet: Antibody suppliers, catalogue numbers and concentrations used were: rabbit anti‐CHMP1a (Proteintech #15761‐1‐AP, 1:500), rabbit anti‐CHMP1b (Proteintech #14639‐1‐AP, 1:500), rabbit anti‐IST1 (Biorad #VPA00314, 1:500), mouse anti‐CHMP5 (Santa Cruz #sc‐374338, 1:500), rabbit anti‐4E‐BP1 (Cell Signaling Technology #9644, rabbit anti‐p‐4E‐BP1‐Ser65 (Cell Signaling Technology #9456, 1:1000), rabbit anti‐S6 (Cell Signaling Technology #2217, 1:4000), rabbit anti‐p‐S6‐Ser240/244 S6 (Cell Signalling Technology #5364, 1:4000), rabbit anti‐Caveolin‐1 (Cell Signaling Technology #3238, 1:500), goat anti‐AREG (R&D Systems #AF262, 1:200), mouse anti‐Tubulin (Sigma #T8328, 1:4000), mouse anti‐CD81 (Santa Cruz #23962, 1:500), mouse anti‐CD63 (BD Biosciences # 556019, 1:500), rabbit anti‐Syntenin‐1 antibody (Abcam ab133267, 1:500), rabbit anti‐Tsg101 (Abcam ab125011, 1:500), mouse anti‐Rab11 (BD Biosciences #610657, 1:500), sheep anti‐TGN46 (BioRad; AHP500G, 1:1000), rabbit anti‐EEA1 (Cell Signalling Technology #3288, 1:1000) , anti‐mouse IgG (H+L) HRP conjugate (Promega #W4021, 1:10000), anti‐rabbit IgG (H+L) HRP conjugate (Promega #W4011, 1:10000), anti‐goat IgG (H+L) HRP conjugate (R&D Systems #HAF109, 1:100).

Techniques: Western Blot, Isolation, Size-exclusion Chromatography, Transduction, shRNA, Construct, Derivative Assay, Activity Assay, Cell Culture, Produced

Journal: bioRxiv

Article Title: A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance

doi: 10.1101/2023.02.26.530079

Figure Lengend Snippet: (A) Viability measurement of C1-treated A549 cells after 48 hours treatment, including effects of treatments with indicated products of the folate-mediated one-carbon metabolism. Data was collected in n=2 biological replicates. (B) Western-blot analysis of the mTORC1 substrate 4E-BP1 in A549 cells after 15.5 hours 100 nM C1-or 100 nM Methotrexate-treatment, including 2.5 rescue treatments with purines. Rescue treatments consisted of 40 μM adenosine, 40 μM guanosine or a combination of 20 μM adenosine and 20 μM guanosine. Reduced mTORC1 activity leads to reduced 4E-BP1 phosphorylation, which is visualized as a mobility shift to a faster-migrating form.

Article Snippet: The following primary antibodies were used for Western blotting (WB) and immunofluorescence (IF): rat anti-HA (Roche, 11867423001), mouse anti-FLAG (Sigma-Aldrich, F3165), rabbit anti-4E-BP1 (Cell Signaling, 9452), mouse anti-TOM20 (BD transduction laboratories, 612278), mouse anti-LAMP1 (BD Pharmingen, 555798), rabbit anti-Akt (Cell Signaling, 9272) and mouse anti-Actin (MP Biomedicals, 691001).

Techniques: Western Blot, Activity Assay, Mobility Shift

Journal: Cell reports

Article Title: A multiplexed in vivo approach to identify driver genes in small cell lung cancer

doi: 10.1016/j.celrep.2023.111990

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used: anti-HSP90 (CST 4877, 1:4,000), anti-TSC1 (D43E2, CST 6935, 1:200), anti-TSC2 (D93F12, CST 4308), anti-S6 (CST 2217, 1:200), anti-phospho-S6 (Ser235/236, CST 2211, 1:200), anti-GFP (D5.1, CST 2956, 1:200), anti-p-mTOR (D9C2, Ser2448, CST 55365, 1:200), anti-mTOR (CST 2972, 1:200), anti-phospho-4E-BP1 (Ser65, CST 9451, 1:200), and anti-4E-BP1 (53H11, CST 9644, 1:200).

Techniques: Plasmid Preparation, Recombinant, Modification, Labeling, Amplification, Staining, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, DNA Extraction, Purification, Sequencing, Generated, Software

Journal: Cell reports

Article Title: A multiplexed in vivo approach to identify driver genes in small cell lung cancer

doi: 10.1016/j.celrep.2023.111990

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-4E-BP1 Monoclonal , Cell Signaling Technology , Cat#9644; RRID:AB_2097841.

Techniques: Plasmid Preparation, Recombinant, Modification, Labeling, Amplification, Staining, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, DNA Extraction, Purification, Sequencing, Generated, Software

Journal: Cell reports

Article Title: A multiplexed in vivo approach to identify driver genes in small cell lung cancer

doi: 10.1016/j.celrep.2023.111990

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-phospho-4E-BP1 (Ser65) Polyclonal , Cell Signaling Technology , Cat#9451; RRID:AB_330947.

Techniques: Plasmid Preparation, Recombinant, Modification, Labeling, Amplification, Staining, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, DNA Extraction, Purification, Sequencing, Generated, Software

Journal: JCI Insight

Article Title: TFEB-mediated lysosomal exocytosis alleviates high-fat diet–induced lipotoxicity in the kidney

doi: 10.1172/jci.insight.162498

Figure Lengend Snippet: ( A ) Representative Western blot images of phosphorylated (p-) TFEB S211, total TFEB, p-S6RP (Ser235/236), S6RP, p–4E-BP1 (Thr37/46), and 4E-BP1 in cultured PTECs after Torin 1, BSA, or PA treatment for 6 hours ( n = 3). ( B ) Representative Western blot images of p-S6RP (Ser235/236), S6RP, p-4E-BP1 (Thr37/46), and 4E-BP1 in cultured PTECs subjected to BSA or PA treatment for the indicated periods ( n = 3). The values are normalized by the value at time 0. ( C ) Representative immunofluorescence images of TFEB (green) in cultured PTECs transfected with a constitutively active form of HA-tagged RagC for 48 hours, including treatment with Torin 1, BSA, or PA for the last 6 hours ( n = 3). Cells were immunostained for HA (red) and counterstained with DAPI (blue). The percentage of PTECs exhibiting TFEB nuclear translocation was determined in wild-type PTECs (RagC − ) and PTECs transfected with HA-tagged RagC (RagC + ). ( D ) Representative Western blot images of TFEB, p-S6RP (Ser235/236), S6RP, p-4E-BP1 (Thr37/46), and 4E-BP1 in cultured PTECs either starved of amino acids for 60 minutes or starved for 60 minutes and then restimulated with amino acids for 30 minutes after BSA or PA treatment for 6 hours ( n = 3). Bars: 10 μm ( C ). Data are provided as means ± SEM. Statistically significant differences: * P < 0.05 versus RagC − PTECs with the same treatment; # P < 0.05 versus BSA-treated PTECs ( A and B , 2-tailed Student’s t test; C , 1-way ANOVA followed by the Tukey-Kramer test). S6RP, S6 ribosomal protein.

Article Snippet: We used the following antibodies: LRP2/MEGALIN (gifted by T. Michigami, Department of Bone and Mineral Research, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan), TFEB (Bethyl, A303-673A, for mouse samples; Cell Signaling Technology, 37785, for human samples), phospho-TFEB (Ser211; Cell Signaling Technology, 37681), Lamin A/C (Cell Signaling Technology, 2032), GAPDH (GeneTex, GTX100118), S6RP (Cell Signaling Technology, 2217), phospho-S6RP (Ser235/236; Cell Signaling Technology, 2211), 4E-BP1 (Cell Signaling Technology, 9644), phospho-4E-BP1 (Thr37/46; Cell Signaling Technology, 2855), MTOR (Cell Signaling Technology, 2983), ACTB (MilliporeSigma, A5316), HA (MilliporeSigma, 11867423001), FLCN (Cell Signaling Technology, 3697), GABARAP (MBL, PM037), LAMP1 (BD Biosciences, 553792 for mouse samples; BD Biosciences, 555798 for human samples), CTSD (Santa Cruz Biotechnology, sc-6486), TMEM55B (Proteintech, 23992-1-AP), MAP1LC3B (Cell Signaling Technology, 2775), COL1A1 (Abcam, ab34710), F4/80 (Bio-Rad, MCA497), GALECTIN-3 (Santa Cruz Biotechnology, sc-23938), SQSTM1/p62 (PROGEN, GP62-C), biotinylated secondary antibodies (Vector Laboratories, BA-1000, BA-2001, BA-4000, and BA-7000), horseradish peroxidase–conjugated secondary antibodies (DAKO, P0447, P0448, P0449, and P0450), and Alexa Fluor–conjugated secondary antibodies (Thermo Fisher Scientific, A21206, A21208, A21434, and A31572).

Techniques: Western Blot, Cell Culture, Immunofluorescence, Transfection, Translocation Assay