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

Journal: Neuron

Article Title: Bassoon Controls Presynaptic Autophagy through Atg5.

doi: 10.1016/j.neuron.2017.01.026

Figure Lengend Snippet: Figure 3. Autophagosomes Accumulate in Periaxonal Soma of DKD Neurons (A) Images of DIV14 mCh-Lamp1/SC (top) and mCh-Lamp1/DKD (bottom) neurons immunostained with Ankyrin-G (arrows) to mark the initial segment of the axon. mCh-Lamp1 puncta are clustered in the periaxonal soma of the DKD neuron. (B) Quantitation of Lamp1 asymmetry in SC and DKD neurons shows an increase in axon-proximal mCh-Lamp1 in DKD neurons. (C) Images of SC and DKD neuronal cell soma (DIV14) labeled with Lysotracker reveals increased axon-proximally located lysosomes in DKD neuron. (D) Quantitation of axon-proximal versus axon-distal Lysotracker puncta in EGFP SC- and EGFP DKD-expressing neurons is shown. (E) Mature autolysosomes, identified as mRFP+/EGFP puncta, were asymmetrically distributed in the soma of tf-LC3 and DKD neurons (DIV14). The addition of 100 nM bafilomycin to DKD neurons (lower panel) increased the number of EGFP+ puncta in DKD neurons. (F) Quantitation of fluorescent puncta from tf-LC3 and DKD neurons treated with DMSO or DMSO and 100 nM bafilomycin A for 3 hr shows a marked increase in mRFP+/EGFP+ puncta after bafilomycin administration. (G) EM micrograph of the cell soma of a DIV14 VAMP2-HRP/DKD neuron containing DAB-labeled vesicular structures as cargo in an autophagic vacuole-like structure. N marks the cell nucleus. DAB-labeled structures were not observed in the soma of any VAMP2-HRP/SC neurons. Scale bars, 10 mm (A, C, and G) and 500 nm (G). Graphs represent mean ± SEM. See Table S1 for means ± SEM, scoring, and n and p values. See also Figure S3 with Movies S1 and S2 and Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human: HeLa, Passage < 30 ATCC CRL-7923 Experimental Models: Organisms/Strains Wildtype rats: Sprague Dawley Charles River, Harlan N/A Wildtype mice: C57Bl6/N Research Institutes for experimental Medicine (FEM) Charité Berlin N/A Bassoon knockout mice Leibniz Institute for Neurobiology Magdeburg: laboratory of Eckart D. Gundelfinger (Davydova et al., 2014) Omnibank ES cell line OST486029 by Lexicon Pharmaceuticals (The Woodlands, TX, USA) Lentivirus Garner and Reimer labs, Stanford University; Virus Core Facility, Charité Berlin N/A Helper-dependent adenovirus (HdAd) Philip Ng, Baylor College of Medicine; (Palmer & Ng 2011) N/A Recombinant DNA FU-SV2-eGFP DKD/SC-W Waites et al., 2013 N/A FU-Vamp2-HRP DKD/SC-W Waites et al., 2013 N/A FU-SV2-eGFP BsnKD-W Waites et al., 2013 N/A FU-eGFP DKD/SC-Wm Waites et al., 2013 N/A FU-mCherry-HA-Ubiquitin K0-W Waites et al., 2013 N/A FU-eGFP-Synapsin1a-W Leal-Ortiz et al., 2008 N/A FU-Synapsin1a Siah1KD-Wm This paper N/A pBsnCC2-eGFP (aa2087-2562, CAA76287.1) Maas et al., 2012 N/A pBsnZF-eGFP (aa1-609, CAA76287.1) Maas et al., 2012 N/A pBsnCD-eGFP(aa1691-3263, CAA76287.1) Maas et al., 2012 N/A pBsnFL-eGFP (aa95-3938, CAA76287.1) Maas et al., 2012 N/A FU-tf-LC3 DKD/SC-Wm This paper ptf-LC3 from Addgene #21073 FU-mRFP-LC3 DKD/SC-Wm This paper mRFP-LC3 from Addgene #21075 FU-mCh-Atg5 DKD/SC-Wm This paper mCh-Atg5 from Addgene #13095 FU-mCh-Lamp1 DKD/SC-Wm This paper pSPAX2 from Addgene #12260 FU-tf-LC3 –Wm This paper ptf-LC3 from Addgene #21073 FU-mRFP-LC3 –Wm This paper mRFP-LC3 from Addgene #21075 FU-mCh-Atg5 –Wm This paper mCh-Atg5 from Addgene #13095 FU-eGFP shAtg5-Wm This paper N/A cDNA rat Lamp1 (NM_012857.2) This paper N/A Lamp1-mCherry This paper pmCherry from Clontech Cat# 632524 FU CFP-SV2 DKD/SC-Wm Noam E. Ziv, Technion, Institute of Technology Haifa Israel N/A FU-CC2v1-eGFP-Synapsin1a-W This paper N/A FU-CC2v1*-eGFP-Synapsin1a-W This paper N/A mRFP-Bassoon* FL Waites et al., 2013 N/A FU-mCherry-Wm This paper N/A pEGFP-N Clontech Cat# 632469 pCMV-VSVg Generous gift of Dr. Theo Palmer; Stanford University N/A Sequence-Based Reagents Atg5 shRNA Obtained from the Stanford Protein and Nucleic Acid facility according to Lee and Gao, 2009 N/A fw 50-gatccccatctgagctatccagacaattcaagag attgtctggatagctcagattttttggaaa-30 Obtained from the Stanford Protein and Nucleic Acid facility according to Lee and Gao, 2009 N/A (Continued on next page) Neuron 93, 897–913.e1–e7, February 22, 2017 e2

Techniques: Quantitation Assay, Labeling, Expressing

Journal: iScience

Article Title: NRF3 activates mTORC1 arginine-dependently for cancer cell viability

doi: 10.1016/j.isci.2023.106045

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal LAMP-1 (H4A3) antibody , Santa Cruz Biotechnology , Cat# sc-20011; RRID:AB_626853.

Techniques: Recombinant, ATP Assay, Sequencing, Software

Journal: Nature Communications

Article Title: Cascaded immunotherapy with implantable dual-drug depots sequentially releasing STING agonists and apoptosis inducers

doi: 10.1038/s41467-025-56407-7

Figure Lengend Snippet: a – f Transcriptomic analysis based on RNA sequencing of B16F10 tumors in the MS-DF-MS group and control group subjected to the treatment for 12 h ( n = 3 mice). a Upset plot with Venn diagram illustrating the gene count and overlap across samples. b Interactive heatmap and clustering analysis of DEGs ( P < 0.05 and FC > 2 or FC < 0.5). c Volcano plot illustrating significantly upregulated ( P < 0.05 and FC > 2) and significantly downregulated ( P < 0.05 and FC < 0.5) genes (MS-DF-MS group versus control group) and the GOI in DEGs. Heatmap displaying expression variations ( d ), circos plot with pathway enrichments ( e ), and PPI network ( f ) among the GOI in significantly regulated DEGs. Representative immunofluorescence images ( g ), fluorescence colocalization profiles with correlation coefficients ( R pearson ) ( h ), and quantifications of R pearson ( i ) for STING and lysosome (LAMP1) in B16F10 cells with indicated treatments for 6 h. M1, RAW264.7 cells undergoing co-incubation (24 h) with B16F10 cell debris generated by the treatment (24 h) with MS-DF-MS ( n = 3 independent experiments). j – l Representative flow cytometry half-overlapped histograms ( j ) and quantifications ( k ) of STING + fluorescence, and representative immunofluorescence images of microtubules (α-tubulin) ( l ) in B16F10 cells with indicated treatments ( n = 3 independent experiments). m Schematic diagram exhibiting mechanisms of the STING signaling enhancement by activating NF-κB to depolymerize microtubules, suppress the STING trafficking into lysosomes, and prevent the STING degradation in cancer cells, dendritic cells, or macrophages. Data are expressed as mean ± SD. Statistical significances were assessed using unpaired two-tailed Student’s t test in a – f and one-way ANOVA with Tukey’s multiple comparisons post hoc test in i and k . Source data are provided as a Source Data file.

Article Snippet: Rabbit-derived anti-GAPDH polyclonal antibody (#10494-1-AP), mouse-derived anti-vinculin monoclonal antibody (#66305-1-Ig), and rat-derived anti-mouse LAMP1 monoclonal antibody labeled with APC (#APC-65050) were obtained from Proteintech (Wuhan, China).

Techniques: RNA Sequencing, Control, Expressing, Immunofluorescence, Fluorescence, Incubation, Generated, Flow Cytometry, Two Tailed Test