Journal: bioRxiv

Article Title: FICD deficiency protects mice from hypertrophy-induced heart failure via BiP-mediated activation of the UPR ER and ER-phagy

doi: 10.1101/2024.05.28.596287

Figure Lengend Snippet: Hearts were collected from 8-month mice 8 weeks post-TAC or sham surgery and processed for protein quantification. Protein from (A) sham only, (B) TAC only, (C) WT only, and (D) Ficd -/- only mice were run on 15% Tris-Glycine SDS acrylamide gels, blocked with 5% BSA, and probed for LC3 and GAPDH (loading control) levels (n=6-7 mice per genotype per condition). Quantification of LC3-I, LC3-II, and GAPDH from (E) sham only, (F) TAC only, (G) WT only, (H) Ficd -/- only gels. Protein from (I) sham only and (J) TAC only mice were run on 10% Tris-Glycine SDS acrylamide gels, blocked with 5% BSA, and probed for CtsL, CtsB, CtsD, LAMP1, and GAPDH (loading control) levels (n=6-7 mice per genotype per condition). Quantification of (K) CtsL, (L), CtsB, (M) CtsD, and (N) LAMP1 levels relative to GAPDH controls in sham only mice. Quantification of (O) CtsL, (P), CtsB, (Q) CtsD, and (R) LAMP1 levels relative to GAPDH controls in TAC only mice. Indicated P values were calculated using unpaired t tests.

Article Snippet: The following antibodies were used for western blotting and immunofluorescence studies: Alpha-actinin (D6F6)(Cell Signaling, 6487T, Rabbit mAb), BiP (Proteintech, 665741IG, mouse mAb), CHOP (D46F1)(Cell Signaling, 5554S, Rabbit mAb), Cathepsin B (D1C7Y)(Cell Signaling, 31718S, Rabbit mAb), Cathepsin D (E179)(Cell Signaling, 69854S, Rabbit mAb), Cathepsin L (Santa Cruz Biotechnology, sc-32320, mouse mAb), FAM134B (E8Y9R)(Cell Signaling, 83414S, Rabbit mAb), GAPDH (Proteintech, 600041IG, mouse mAb), HSC70 (B-6)(Santa Cruz Biotechnology, sc-7298, mAb), LAMP1 (E6N3R)(Cell Signaling, 46843S, Rabbit mAb), LC3A/B (D3U4C)(Cell Signaling, 12741S, Rabbit mAb), Thr-AMPylation (Biointron, 17G6, mAb), Secondary 647 Fluorescent antibody (ThermoFisher, A32728).

Techniques:

Journal: bioRxiv

Article Title: FICD deficiency protects mice from hypertrophy-induced heart failure via BiP-mediated activation of the UPR ER and ER-phagy

doi: 10.1101/2024.05.28.596287

Figure Lengend Snippet: (A) Primary WT and Ficd -/- cardiomyocytes were isolated from 6-8 mice hearts per condition and plated in 24-well dishes for 48 hours at 37°C and 5% CO2. Cardiomyocytes were treated with Tm (10μg/mL) or Tg (3μM) for 10 hours and supplemented with bafilomycin (100nM) for four hours before being collected for protein quantification via western blotting. Cells were run on 15% Tris-Glycine SDS acrylamide gels, blocked with 5% BSA, and probed for LC3, FAM134B, and HSC70 (loading control) levels. Levels of (B) LC3-II/HSC70, (C) LC3-II/LC3-I, and (D) FAM134B/HSC70 were quantified from control, ER-stressed, and bafilomycin treated cardiomyocytes. (E) Primary WT and Ficd -/- cardiomyocytes were isolated from 6-8 mice hearts per condition and plated in 6-well dishes for 48 hours at 37°C 5% CO2. Cardiomyocytes were treated with Tm (10μg/mL) or Tg (3μM) for 12 hours before being collected for protein quantification via western blotting. Cells were run on 10% Tris-Glycine SDS acrylamide gels, blocked with 5% BSA, and probed for CtsL, CtsB, CtsD, LAMP1, and HSC70 (loading control) levels. Levels of (F) CtsL/HSC70, (H) CtsB/LC3-I, (I) CtsD/HSC70, and (J) LAMP1/HSC70 were quantified from control and ER-stressed cardiomyocytes. (G) CtsL activity from 50μg of isolated WT and Ficd -/- cardiomyocytes from non-treated and CtsL inhibitor treated cells. (K) Transmission Electron Microscopy images of ER whorls from isolated WT and Ficd -/- cardiomyocytes. (Scale bar, 200nm). (L) Co-immunoprecipitation of FAM134B-BiP from control, Tg-treated, and FICD(E324G) transfected WT and Ficd -/- cardiomyocytes. FAM134B antibody was incubated with 150μg of protein from each cardiomyocyte condition and immunoprecipitated with Protein A Magnetic agarose beads. 10% input and the IP-ed proteins were run on 10% Tris-Glycine acrylamide gels, blocked with 5% BSA, and probed for Thr-AMPylation, BiP, FAM134B, and HSC70 (loading control). (M) Quantification of three replicate co-immunoprecipitations of BiP/FAM134B from control, Tg, and FICD(E324G) transfected WT and Ficd -/- cardiomyocytes. Indicated P values were calculated using unpaired t tests. 2-way ANOVA tests were performed in parallel to confirm significance of results.

Article Snippet: The following antibodies were used for western blotting and immunofluorescence studies: Alpha-actinin (D6F6)(Cell Signaling, 6487T, Rabbit mAb), BiP (Proteintech, 665741IG, mouse mAb), CHOP (D46F1)(Cell Signaling, 5554S, Rabbit mAb), Cathepsin B (D1C7Y)(Cell Signaling, 31718S, Rabbit mAb), Cathepsin D (E179)(Cell Signaling, 69854S, Rabbit mAb), Cathepsin L (Santa Cruz Biotechnology, sc-32320, mouse mAb), FAM134B (E8Y9R)(Cell Signaling, 83414S, Rabbit mAb), GAPDH (Proteintech, 600041IG, mouse mAb), HSC70 (B-6)(Santa Cruz Biotechnology, sc-7298, mAb), LAMP1 (E6N3R)(Cell Signaling, 46843S, Rabbit mAb), LC3A/B (D3U4C)(Cell Signaling, 12741S, Rabbit mAb), Thr-AMPylation (Biointron, 17G6, mAb), Secondary 647 Fluorescent antibody (ThermoFisher, A32728).

Techniques: Isolation, Western Blot, Activity Assay, Transmission Assay, Electron Microscopy, Immunoprecipitation, Transfection, Incubation

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Pyrrolidine SS13 Induces Oxidative Stress and Autophagy-Mediated Cell Death in Colorectal Cancer Cells.

doi: 10.1016/j.ejps.2024.106982

Figure Lengend Snippet: Figure 11. Immunofluorescence staining of LAMP1 and LysoTracker in HCT116 (A) and Caco-

Article Snippet: LAMP1 (D2D11) XP® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and LC3B (E5Q2K) Mouse mAb (Cell Signaling Technology, Danvers, MA, USA) primary antibodies were used on the cells for 90 min at room temperature.

Techniques: Immunofluorescence, Staining

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Pyrrolidine SS13 Induces Oxidative Stress and Autophagy-Mediated Cell Death in Colorectal Cancer Cells.

doi: 10.1016/j.ejps.2024.106982

Figure Lengend Snippet: Figure 14. Immunofluorescence staining of LAMP1 and LC3B in HCT116 after SS13 (7 µM),

Article Snippet: LAMP1 (D2D11) XP® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and LC3B (E5Q2K) Mouse mAb (Cell Signaling Technology, Danvers, MA, USA) primary antibodies were used on the cells for 90 min at room temperature.

Techniques: Immunofluorescence, Staining

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Pyrrolidine SS13 Induces Oxidative Stress and Autophagy-Mediated Cell Death in Colorectal Cancer Cells.

doi: 10.1016/j.ejps.2024.106982

Figure Lengend Snippet: Figure 15. Immunofluorescence staining of LAMP1 and LC3B in Caco-2 cells treated with SS13

Article Snippet: LAMP1 (D2D11) XP® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and LC3B (E5Q2K) Mouse mAb (Cell Signaling Technology, Danvers, MA, USA) primary antibodies were used on the cells for 90 min at room temperature.

Techniques: Immunofluorescence, Staining

Journal: Nature

Article Title: A phosphoinositide signalling pathway mediates rapid lysosomal repair.

doi: 10.1038/s41586-022-05164-4

Figure Lengend Snippet: Fig. 2 | PtdIns4P drives ORP-tethered ER–lysosomal contacts for rapid membrane repair. a, ORP family members are enriched on damaged lysosomes. Note that ORP10 is absent from S20 (supernatant after centrifugation at 20,000 g) (arrow head). Asterisk indicates a nonspecific band. b, Endogenous ORP9 is recruited to LAMP1-positive lysosomes upon brief LLOME treatment in U2OS cells. c, Punctate intensities of endogenous ORP9 and ORP11 before and after LLOME treatment. More than 30 random cells were quantified for each condition. Data are mean ± s.e.m.; n = 4 trials. See fluorescence images in Extended Data Fig. 5m. d, LLOME induces extensive wrapping of endogenous LAMP1 by eGFP–VAPA in U2OS cells. Right, schematic illustration of the process. e, Percentage of U2OS cells with more than five lysosomes extensively wrapped by eGFP–VAPA. More than 100 cells were counted for each condition. Data are mean ± s.e.m.; n = 3 per condition. See fluorescence images in Extended Data Fig. 6a. f, Representative microscopy and quantification of ER–lysosome wrapping in various ORP-knockout cells. More than 100 cells were counted for each condition. Data are mean ± s.e.m.; n = 3. See more details in Extended Data Fig. 6c–f.

Article Snippet: To generate the Lyso-TurboID construct (pCDH-LAMP1-mGFP-TurboID), the LAMP1– mGFP sequence was amplified from Addgene Plasmid 34831 and fused with TurboID sequence into pCDH-CMV-MCS.

Techniques: Membrane, Centrifugation, Fluorescence, Microscopy, Knock-Out

Journal: Nature

Article Title: A phosphoinositide signalling pathway mediates rapid lysosomal repair.

doi: 10.1038/s41586-022-05164-4

Figure Lengend Snippet: Fig. 3 | ORP9, ORP10 and ORP11 mediate rapid lysosomal repair by ER-to-lysosome transfer of phosphatidylserine. a, Schematic of the in vitro lipid transport assay for ORP9 and ORP11. Note that nitrobenzoxadiazole (NBD) is attached to one fatty acid chain but not the head group of PS. PE, phosphatidylethanolamine; Rh, rhodamine; DGS–NTA(Ni), a nickel-chelating lipid that binds His-tagged proteins. b, Lipid transfer assay demonstrating the increase in NBD fluorescence stimulated by ORP9 and ORP11 in the presence of NBD–PS but not NBD–cholesterol (Chol) or TopFluor (TF)-cholesterol. Data are mean ± s.e.m.; n = 3 per condition. c, U2OS cells stably expressing the PS probe GFP–Lact-C2 were treated with LLOME for 20 min and washed with detergent before immunostaining of endogenous IST1 and LAMP1. Scale bars, 10 μm. d, Colocalization of GFP–Lact-C2 and LAMP1 in the indicated U2OS cell lines. Data are mean ± s.e.m. of Pearson’s correlation coefficient; n = 30 cells from 3 trials per condition. See fluorescence images in Extended Data Fig. 7f. e, Schematic illustration of different ORP mutants used in f,h. f, U2OS cells stably expressing GFP–Lact-C2 and indicated mCherry–ORP9 and ORP11 proteins (mutants are notated as ORP9 mutant/ORP11 mutant) were treated as in c, and the Pearson’s

Article Snippet: To generate the Lyso-TurboID construct (pCDH-LAMP1-mGFP-TurboID), the LAMP1– mGFP sequence was amplified from Addgene Plasmid 34831 and fused with TurboID sequence into pCDH-CMV-MCS.

Techniques: In Vitro, Transport Assay, Fluorescence, Stable Transfection, Expressing, Immunostaining, Mutagenesis

Journal: Nature

Article Title: A phosphoinositide signalling pathway mediates rapid lysosomal repair.

doi: 10.1038/s41586-022-05164-4

Figure Lengend Snippet: Fig. 4 | PS-activated lipid transport by ATG2 mediates direct, rapid lysosomal repair. a, Live-cell imaging demonstrating rapid recruitment of eGFP–ATG2A to damaged lysosomes in U2OS cells stably expressing eGFP– ATG2A and LAMP1–mCherry. Scale bar, 10 μm. b, Quantification of eGFP–ATG2A puncta in individual U2OS cells upon LLOME treatment. c, eGFP–galectin-3 assay showing defects of rapid lysosomal repair in four independent clones of ATG2A/B-DKO cells. Data are mean ± s.e.m.; 50–100 cells, n = 3 for each condition. See more details in Extended Data Fig. 9a,b. d, Experimental design for the reconstitution of PS-stimulated lipid transport by ATG2A using a FRET-based assay. e, PS in acceptor liposomes potently stimulates ATG2A-dependent NBD fluorescence. Data are mean ± s.e.m.; n = 3 per condition. f, Illustration of lipid transport mutants of ATG2A. Flexible loops outside of the main structure are not shown. g, In vitro lipid transport assays testing the activity of various ATG2A mutants. Data are mean ± s.e.m.; n = 3 per condition. h, eGFP–galectin-3 assay demonstrating that ATG2A lipid transport

Article Snippet: To generate the Lyso-TurboID construct (pCDH-LAMP1-mGFP-TurboID), the LAMP1– mGFP sequence was amplified from Addgene Plasmid 34831 and fused with TurboID sequence into pCDH-CMV-MCS.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Clone Assay, Liposomes, Fluorescence, In Vitro, Activity Assay