Journal: Cell reports

Article Title: Ku proteins promote DNA binding and condensation of cyclic GMP-AMP synthase.

doi: 10.1016/j.celrep.2022.111310

Figure Lengend Snippet: Figure 4. Ku proteins augment cGAS cata- lytic activity by promoting cGAS-DNA binding (A and B) Ku80- (A) or Ku70- (B) knockdown and control THP-1 cells were stimulated with HT-DNA (1 mg/mL) for 6 h. The cells were harvested for cGAMP measurement by ELISA (top) and immu- noblotting assay (bottom). (C) Recombinant cGAS (0.5 mM) and GFP (4 mM), Ku80 (0.9 mM), or Ku70 (0.9 or 4 mM) were incubated with HT-DNA (0.2 mg/mL). cGAMP production was measured by ELISA. (D) Ku80- or Ku70-knockdown and control THP-1 cells were transfected with biotin-ISD (1 mg/mL) for 2 h. The cell lysates were incubated with streptavi- din-Sepharose beads for 4 h and analyzed by immunoprecipitation and immunoblotting. (E) Similar to (D), except wild-type and Ku80-defi- cient HeLa cells were used. (F) Recombinant cGAS protein (3.4 nM) together with GFP (100 nM), Ku80 (5 nM), or Ku70 (5 or 100 nM) was incubated with streptavidin-Sephar- ose beads with or without biotin-ISD (1 mg) for 3 h and then pulled down with streptavidin-Sepharose beads and analyzed by immunoblotting. (G) Recombinant GFP (5.5 nM) or Ku80 (2.75 or 5.5 nM) was incubated with or without cGAS (100 nM) in the presence of Cy5-ISD (1.25 mM) and analyzed by EMSA to measure cGAS-DNA binding ability. Data shown in (A–C) are from one representative experiment of at least three independent experi- ments (mean ± SD, n = 2 biological replicates). *p < 0.05, **p < 0.01, two-tailed Student’s t test. See also Figure S6.

Article Snippet: HEK293T ATCC CRL-11268 HEK293A Hongyu Deng, Institute of Biophysics, Chinese Academy of Sciences N/A HeLa ATCC CCL-2 Vero ATCC CCL-81 immortalized MEF This paper N/A HEK293T stably expressing Flag-STING This paper N/A Ku80 deficiency THP-1 This paper N/A Ku70 deficiency THP-1 This paper N/A DNA-PKcs knockout THP-1 This paper N/A DNA-PKcs knockout HEK293T This paper N/A cGAS knockout THP-1 This paper N/A STING knockout THP-1 This paper N/A Oligonucleotides ISD:sense 50-TACAGATCTACTAGTG ATCTATGACTGATCTGTACATGATCTACA-30 This paper N/A sgRNA and shRNA, see Table S2 This paper N/A primers for qPCR, see Table S3 This paper N/A Recombinant DNA IFNb-Luc Hongbing Shu, Wuhan University N/A ISRE-Luc Hongbing Shu, Wuhan University N/A NF-kB-Luc Zhijian Chen, University of Texas, Southwestern Medical Center at Dallas N/A Other recombinant DNA used in this study are listed in Table S1 This paper N/A Software and algorithms GraphPad Prism 8 GraphPad https://www.graphpad.com/guides/prism/ 8/user-guide/tips_for_using_prism.htm ImageJ NIH https://imagej.nih.gov/ij/download.html NIS-Elements AR Analysis 5.20.00 NIKON https://www.microscope.healthcare. nikon.com/products/software/ nis-elements Imaris 9.5.1 IMARIS https://imaris.oxinst.com/ Cell Reports 40, 111310, September 6, 2022 e2

Techniques: Activity Assay, Binding Assay, Knockdown, Control, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Transfection, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Chronic treatment with the complex I inhibitor MPP + depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2–associated athanogene 6 (BAG6)

doi: 10.1074/jbc.RA119.010474

Figure Lengend Snippet: Chronic MPP+ treatment induces BAG6 expression. Differentiated SH-SY5Y cells were either untreated (Ctrl) or treated with 250 μm of MPP+ for 2 weeks, and protein expression was analyzed. A, chronic MPP+ treatment induces BAG6 expression and decreases BAG2, BAG5, and PINK1 levels. B, BAG5 protein quantification. *, p = 0.0005. C, BAG2 protein quantification. *, p < 0.0001. D, BAG6 protein quantification. *, p < 0.0001; see also Fig. 1F. E, PINK1-FL. *, p < 0.0001 and PINK1-dN protein quantification, *, p < 0.0001. F, BAG6 mRNA expression normalized to GAPDH, p = 0.0037. Graphs represent means ± S.D.; n = 5 independent experiments for B–E; n = 4 independent experiments for F; Student's two-tailed t test.

Article Snippet: Densitometry data were normalized to loading control, and the half-lives of PINK1 isoforms were estimated by fitting a one-phase exponential decay curve using Prism 8 software (GraphPad, San Diego, CA). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Catalog number Source Dilution PINK1 Rabbit BC100-494 Novus Biological 1:1000 TOM20 Rabbit Sc11415 Santa Cruz 1:10,000 β-ACTIN Mouse A5316 Sigma 1:2000 HSP60 Mouse 611563 BD Transduction 1:1000 BAG6 Mouse Sc365928 Santa Cruz 1:1000 BAG5 Rabbit NB100-56091 Novus Biological 1:1000 BAG2 Mouse Sc101216 Santa Cruz 1:1000 HSP90 Rabbit 4877 Cell Signaling 1:1000 CDC37 Rabbit 3604 Cell Signaling 1:1000 CHIP Rabbit Sc66830 Santa Cruz 1:1000 GAPDH Rabbit Ab37168 Abcam 1:10,000 GFP Rabbit A6455 Invitrogen 1:10,000 V5 Mouse Sc271944 Santa Cruz 1:1000 FLAG Rabbit F7425 Sigma 1:1000 β-Tubulin Rabbit Ab6046 Abcam 1:5000 Open in a separate window List of antibodies used in the study

Techniques: Expressing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Chronic treatment with the complex I inhibitor MPP + depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2–associated athanogene 6 (BAG6)

doi: 10.1074/jbc.RA119.010474

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Densitometry data were normalized to loading control, and the half-lives of PINK1 isoforms were estimated by fitting a one-phase exponential decay curve using Prism 8 software (GraphPad, San Diego, CA). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Catalog number Source Dilution PINK1 Rabbit BC100-494 Novus Biological 1:1000 TOM20 Rabbit Sc11415 Santa Cruz 1:10,000 β-ACTIN Mouse A5316 Sigma 1:2000 HSP60 Mouse 611563 BD Transduction 1:1000 BAG6 Mouse Sc365928 Santa Cruz 1:1000 BAG5 Rabbit NB100-56091 Novus Biological 1:1000 BAG2 Mouse Sc101216 Santa Cruz 1:1000 HSP90 Rabbit 4877 Cell Signaling 1:1000 CDC37 Rabbit 3604 Cell Signaling 1:1000 CHIP Rabbit Sc66830 Santa Cruz 1:1000 GAPDH Rabbit Ab37168 Abcam 1:10,000 GFP Rabbit A6455 Invitrogen 1:10,000 V5 Mouse Sc271944 Santa Cruz 1:1000 FLAG Rabbit F7425 Sigma 1:1000 β-Tubulin Rabbit Ab6046 Abcam 1:5000 Open in a separate window List of antibodies used in the study

Techniques: Transduction

Journal: Cell reports

Article Title: Medulloblastoma Arises from the Persistence of a Rare and Transient Sox2 + Granule Neuron Precursor.

doi: 10.1016/j.celrep.2020.03.075

Figure Lengend Snippet: Figure 3. Sox2+ Overlaps with Markers of GNPs in the Postnatal EGL (A and B) Rare overlapping GFP and DCX expression was observed in the P0 EGL (A, arrowhead), while no overlap was seen between GFP and NeuN (B). Scale bars, 20 mm. (C) All GFP+ external EGL cells were Pax6+ (arrowheads). Scale bars, 20 mm. (D) Representative IF images of endogenous Sox2 protein and EdU at 3 h post-injection. Scale bar, 20 mm. (E) Quantification of EdU incorporation (in D) in Sox2+ and Sox2 cells (p = 0.0166). n = 5; error bars denote mean ± SEM; two-tailed paired t test. (F) Lineage-tracing strategy in Atoh1Cre ; R26tdT mice. (G) Representative IF images of endogenous Sox2 protein and tdT reporter expression in the P0 cerebellum. Scale bar, 100 mm. (H) Magnified image from (G) illustrating heterogeneous Sox2 expression with clear Atoh1 lineage+ (white arrowheads) and Atoh1 lineage (yellow arrowheads) origins. Scale bars, 20 mm. (I) Atoh1cre ; R26tdT ; Sox2eGFP transgenic mice were bred for fluorescence-activated cell sorting (FACS) analysis of Sox2+ cell lineage origins. (J) Representative FACS plot indicating typical population distribution of tdT and GFP expression from the P0 hindbrain (left). Histogram of tdT+ cells showing percentage of Sox2+ cells derived from the Atoh1 lineage (right).

Article Snippet: ImmPRESS Kit Vector Laboratories Cat# MP-2400, RRID:AB_2336832 ImmPRESS Anti-Rabbit Ig Reagent antibody Vector Laboratories Cat# MP-7401, RRID:AB_2336529 Click-iT Plus EdU Alexa Fluor 647 Imaging Kit ThermoFisher Cat# C10640 Deposited Data Filtered scRNaseq gene matrix from the P0 and P7 murine hindbrain Vladoiu et al., 2019 GEO: GSE118068 Experimental Models: Organisms/Strains Sox2eGFP (Sox2tm1Lpev) Provided by Dr. Freda Miller; Ellis et al., 2004 N/A Sox2CreER (B6;129S-Sox2tm1(cre/ERT2)Hoch/J) Provided by Dr. Konrad Hochedlinger; Arnold et al., 2011 JAX: 017593 R26tdT (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) The Jackson Laboratory; Madisen et al., 2010 JAX: 007914 Ptc1LacZ (Ptch1tm1Mps/J) The Jackson Laboratory; Goodrich et al., 1997 JAX: 003081 Ptc1LoxP (B6N.129-Ptch1tm1Hahn/J) The Jackson Laboratory; Uhmann et al., 2007 JAX: 012457 Atoh1cre (B6.Cg-Tg(Atoh1-cre)1Bfri/J) Provided by Dr. Norman Rosenblum; Matei et al., 2005 JAX: 011104 WT CD1 (Crl:CD1(ICR)) Charles River Laboratories CRL: 022 Software and Algorithms ImageJ ImageJ RRID:SCR_003070 Prism 7 and Prism 8 GraphPad RRID:SCR_002798 Adobe Photoshop CS5 Adobe Systems RRID:SCR_014199 NIS-Elements Image Acquisition Software Nikon RRID:SCR_014329 Seurat v3.0 Satija Lab RRID:SCR_016341 Enrichment Map v3.1.0 Merico et al., 2010 RRID:SCR_016052 Volocity Image Acquisition Software Perkin Elmer RRID:SCR_002668 (Continued on next page) Cell Reports 31, 107511, April 14, 2020 e1

Techniques: Expressing, Injection, Two Tailed Test, Transgenic Assay, FACS, Derivative Assay

Journal: Cell reports

Article Title: Medulloblastoma Arises from the Persistence of a Rare and Transient Sox2 + Granule Neuron Precursor.

doi: 10.1016/j.celrep.2020.03.075

Figure Lengend Snippet: Figure 1. The EGL Is Derived from Sox2+ Progenitors in the Embryonic RL (A) Expression of Sox2-eGFP transgene fluorescence and endogenous Sox2 protein detected by IF in the medial cerebellum at E12.5. Scale bars, 100 mm (left panel) and 20 mm (remaining panels). Cb, cerebellum; IC, inferior colliculus; IV, fourth ventricle; MB, midbrain; RL, rhombic lip. (B) Pax6 expression marks the cellular output of the RL at E12.5. Scale bar, 20 mm. (C and D) By E14.5, Sox2 expression becomes restricted to the VZ and RL (C), with cells exiting the RL, marked by Pax6 (D), showing heterogeneous Sox2 expression (arrowheads mark Sox2+ cells). EGL, external germinal layer; RP, roof plate. Scale bars as in (A) and (B). (E) Lineage-tracing strategy following TMX induction at E12.5 or E14.5 to Sox2CreER ; R26tdT embryos. (F) Analysis of tdT reporter expression and its co-localization with Pax6 at E14.5 following E12.5 induction at VZ and RL germinal regions. Scale bars, 100 mm (left panel) and 12 mm (remaining panels). (G) Analysis of tdT reporter expression and its co-localization with Pax6 in the cerebellum at E16.5 following E14.5 induction. Scale bars as in (F).

Article Snippet: ImmPRESS Kit Vector Laboratories Cat# MP-2400, RRID:AB_2336832 ImmPRESS Anti-Rabbit Ig Reagent antibody Vector Laboratories Cat# MP-7401, RRID:AB_2336529 Click-iT Plus EdU Alexa Fluor 647 Imaging Kit ThermoFisher Cat# C10640 Deposited Data Filtered scRNaseq gene matrix from the P0 and P7 murine hindbrain Vladoiu et al., 2019 GEO: GSE118068 Experimental Models: Organisms/Strains Sox2eGFP (Sox2tm1Lpev) Provided by Dr. Freda Miller; Ellis et al., 2004 N/A Sox2CreER (B6;129S-Sox2tm1(cre/ERT2)Hoch/J) Provided by Dr. Konrad Hochedlinger; Arnold et al., 2011 JAX: 017593 R26tdT (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) The Jackson Laboratory; Madisen et al., 2010 JAX: 007914 Ptc1LacZ (Ptch1tm1Mps/J) The Jackson Laboratory; Goodrich et al., 1997 JAX: 003081 Ptc1LoxP (B6N.129-Ptch1tm1Hahn/J) The Jackson Laboratory; Uhmann et al., 2007 JAX: 012457 Atoh1cre (B6.Cg-Tg(Atoh1-cre)1Bfri/J) Provided by Dr. Norman Rosenblum; Matei et al., 2005 JAX: 011104 WT CD1 (Crl:CD1(ICR)) Charles River Laboratories CRL: 022 Software and Algorithms ImageJ ImageJ RRID:SCR_003070 Prism 7 and Prism 8 GraphPad RRID:SCR_002798 Adobe Photoshop CS5 Adobe Systems RRID:SCR_014199 NIS-Elements Image Acquisition Software Nikon RRID:SCR_014329 Seurat v3.0 Satija Lab RRID:SCR_016341 Enrichment Map v3.1.0 Merico et al., 2010 RRID:SCR_016052 Volocity Image Acquisition Software Perkin Elmer RRID:SCR_002668 (Continued on next page) Cell Reports 31, 107511, April 14, 2020 e1

Techniques: Derivative Assay, Expressing

Journal: Cell reports

Article Title: Medulloblastoma Arises from the Persistence of a Rare and Transient Sox2 + Granule Neuron Precursor.

doi: 10.1016/j.celrep.2020.03.075

Figure Lengend Snippet: Figure 3. Sox2+ Overlaps with Markers of GNPs in the Postnatal EGL (A and B) Rare overlapping GFP and DCX expression was observed in the P0 EGL (A, arrowhead), while no overlap was seen between GFP and NeuN (B). Scale bars, 20 mm. (C) All GFP+ external EGL cells were Pax6+ (arrowheads). Scale bars, 20 mm. (D) Representative IF images of endogenous Sox2 protein and EdU at 3 h post-injection. Scale bar, 20 mm. (E) Quantification of EdU incorporation (in D) in Sox2+ and Sox2 cells (p = 0.0166). n = 5; error bars denote mean ± SEM; two-tailed paired t test. (F) Lineage-tracing strategy in Atoh1Cre ; R26tdT mice. (G) Representative IF images of endogenous Sox2 protein and tdT reporter expression in the P0 cerebellum. Scale bar, 100 mm. (H) Magnified image from (G) illustrating heterogeneous Sox2 expression with clear Atoh1 lineage+ (white arrowheads) and Atoh1 lineage (yellow arrowheads) origins. Scale bars, 20 mm. (I) Atoh1cre ; R26tdT ; Sox2eGFP transgenic mice were bred for fluorescence-activated cell sorting (FACS) analysis of Sox2+ cell lineage origins. (J) Representative FACS plot indicating typical population distribution of tdT and GFP expression from the P0 hindbrain (left). Histogram of tdT+ cells showing percentage of Sox2+ cells derived from the Atoh1 lineage (right).

Article Snippet: ImmPRESS Kit Vector Laboratories Cat# MP-2400, RRID:AB_2336832 ImmPRESS Anti-Rabbit Ig Reagent antibody Vector Laboratories Cat# MP-7401, RRID:AB_2336529 Click-iT Plus EdU Alexa Fluor 647 Imaging Kit ThermoFisher Cat# C10640 Deposited Data Filtered scRNaseq gene matrix from the P0 and P7 murine hindbrain Vladoiu et al., 2019 GEO: GSE118068 Experimental Models: Organisms/Strains Sox2eGFP (Sox2tm1Lpev) Provided by Dr. Freda Miller; Ellis et al., 2004 N/A Sox2CreER (B6;129S-Sox2tm1(cre/ERT2)Hoch/J) Provided by Dr. Konrad Hochedlinger; Arnold et al., 2011 JAX: 017593 R26tdT (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) The Jackson Laboratory; Madisen et al., 2010 JAX: 007914 Ptc1LacZ (Ptch1tm1Mps/J) The Jackson Laboratory; Goodrich et al., 1997 JAX: 003081 Ptc1LoxP (B6N.129-Ptch1tm1Hahn/J) The Jackson Laboratory; Uhmann et al., 2007 JAX: 012457 Atoh1cre (B6.Cg-Tg(Atoh1-cre)1Bfri/J) Provided by Dr. Norman Rosenblum; Matei et al., 2005 JAX: 011104 WT CD1 (Crl:CD1(ICR)) Charles River Laboratories CRL: 022 Software and Algorithms ImageJ ImageJ RRID:SCR_003070 Prism 7 and Prism 8 GraphPad RRID:SCR_002798 Adobe Photoshop CS5 Adobe Systems RRID:SCR_014199 NIS-Elements Image Acquisition Software Nikon RRID:SCR_014329 Seurat v3.0 Satija Lab RRID:SCR_016341 Enrichment Map v3.1.0 Merico et al., 2010 RRID:SCR_016052 Volocity Image Acquisition Software Perkin Elmer RRID:SCR_002668 (Continued on next page) Cell Reports 31, 107511, April 14, 2020 e1

Techniques: Expressing, Injection, Two Tailed Test, Transgenic Assay, FACS, Derivative Assay

Journal: Cell reports

Article Title: Medulloblastoma Arises from the Persistence of a Rare and Transient Sox2 + Granule Neuron Precursor.

doi: 10.1016/j.celrep.2020.03.075

Figure Lengend Snippet: Figure 5. Sox2+ GNPs Contribute Extensively to the Granule Lineage and to Lesions in Ptc+/ Germline Mutants (A) Strategy for TMX induction in Sox2CreER ; R26tdT pups at P1 followed by analysis of reporter expression. (B) Rare tdT labeling within the EGL (outlined area) at P2 consistent with expression of endogenous Sox2 protein detected by IF (white arrowheads, relatively high Sox2 expression; gray arrowheads, relatively low Sox2 expression). Scale bars, 12 mm. (C and D) Representative IF image of tdT reporter expression in the P2 cerebellum (C) and quantification of the percent tdT+ cells in the EGL (D). Bar denotes mean; n = 3 biological replicates from at least two litters for each data point. Scale bar, 100 mm.

Article Snippet: ImmPRESS Kit Vector Laboratories Cat# MP-2400, RRID:AB_2336832 ImmPRESS Anti-Rabbit Ig Reagent antibody Vector Laboratories Cat# MP-7401, RRID:AB_2336529 Click-iT Plus EdU Alexa Fluor 647 Imaging Kit ThermoFisher Cat# C10640 Deposited Data Filtered scRNaseq gene matrix from the P0 and P7 murine hindbrain Vladoiu et al., 2019 GEO: GSE118068 Experimental Models: Organisms/Strains Sox2eGFP (Sox2tm1Lpev) Provided by Dr. Freda Miller; Ellis et al., 2004 N/A Sox2CreER (B6;129S-Sox2tm1(cre/ERT2)Hoch/J) Provided by Dr. Konrad Hochedlinger; Arnold et al., 2011 JAX: 017593 R26tdT (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) The Jackson Laboratory; Madisen et al., 2010 JAX: 007914 Ptc1LacZ (Ptch1tm1Mps/J) The Jackson Laboratory; Goodrich et al., 1997 JAX: 003081 Ptc1LoxP (B6N.129-Ptch1tm1Hahn/J) The Jackson Laboratory; Uhmann et al., 2007 JAX: 012457 Atoh1cre (B6.Cg-Tg(Atoh1-cre)1Bfri/J) Provided by Dr. Norman Rosenblum; Matei et al., 2005 JAX: 011104 WT CD1 (Crl:CD1(ICR)) Charles River Laboratories CRL: 022 Software and Algorithms ImageJ ImageJ RRID:SCR_003070 Prism 7 and Prism 8 GraphPad RRID:SCR_002798 Adobe Photoshop CS5 Adobe Systems RRID:SCR_014199 NIS-Elements Image Acquisition Software Nikon RRID:SCR_014329 Seurat v3.0 Satija Lab RRID:SCR_016341 Enrichment Map v3.1.0 Merico et al., 2010 RRID:SCR_016052 Volocity Image Acquisition Software Perkin Elmer RRID:SCR_002668 (Continued on next page) Cell Reports 31, 107511, April 14, 2020 e1

Techniques: Expressing, Labeling

Journal: eLife

Article Title: The SWELL1-LRRC8 complex regulates endothelial AKT-eNOS signaling and vascular function

doi: 10.7554/elife.61313

Figure Lengend Snippet: Figure 2. Leucine-rich repeat-containing protein 8a (LRRC8A) regulates PI3K-AKT-endothelial nitric oxide synthase (eNOS), ERK signaling in endothelium. (A) Western blots of LRRC8A, pAkt2, pAkt1, Akt2, Akt1, pErk1/2, Erk1/2, phosphorylated eNOS (p-eNOS), eNOS, pS6K ribosomal protein, S6K ribosomal protein, GAPDH, and ß-actin in Ad-shScr and Ad-shLrrc8a transduced human umbilical vein endothelial cells (HUVECs) under basal conditions. Quantification of LRRC8A/ß-actin (B), p-eNOS/ß-actin, p-eNOS/total eNOS (C), pAkt2/ß-actin, pAkt2/total Akt2 (D), pAkt1/GAPDH, pAkt1/ total Akt1 (E), pERK1/2/GAPDH, pErk1/2/total Erk1/2 (F), pS6 ribosomal protein/GAPDH, and pS6K ribosomal protein/total S6K ribosomal protein (G). N = 6 independent experiments. Significance between the indicated groups in all blots was calculated using a two-tailed unpaired Student’s t-test. Data are shown as mean ± s.e.m. *p<0.05; **p<0.01; ***p<0.001. The online version of this article includes the following source data and figure supplement(s) for figure 2:

Article Snippet: DOI: https://doi.org/10.7554/eLife.61313 14 of 29 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- pS6 ribosomal (rabbit monoclonal) Cell Signaling Cat#5364s, RRID:AB_ 10694233 WB (1:2000) Antibody Anti- GAPDH (rabbit monoclonal) Cell Signaling Cat#5174s, RRID:AB_1062202 WB (1:2000) Antibody Anti-pErk1/2 (rabbit polyclonal) Cell Signaling Cat#9101s, RRID:AB_331772 WB (1:1000) Antibody Anti-Total Erk1/2 (rabbit polyclonal) Cell Signaling Cat#9102s, RRID:AB_330744 WB (1:1000) Antibody Anti-Grb2 (mouse monoclonal) BD Cat#610111s, RRID:AB_397517 WB (1:1000) Antibody Anti-Grb2 (rabbit monoclonal) Santa Cruz Cat#sc-255, RRID:AB_631602 WB (1:1000) Antibody Anti-LRRC8A (rabbit polyclonal) Pacific antibodies Custom made Epitope: QRTKSRIEQGIVD RSE, WB (1:1000), LRRC8A is a glucose sensor regulating ß-cell excitability and systemic glycemia (Kang et al., 2018) Antibody Anti-CD31 (rabbit polyclonal) Thermo Fisher Cat#MA3105, RRID:AB_223592 IF (1:100) Antibody Anti-IgG (normal mouse IgG) Santa Cruz Cat#sc-2027, RRID:AB_737197 WB (1:1000) Antibody Anti-rabbit-HRP BioRad Cat#170–6515, RRID:AB_11125142 WB (1:10,000) Antibody Anti-mouse-HRP BioRad Cat#170–5047, RRID:AB_11125753 WB (1:10,000) Transfected construct (human) siLrrc8a Invitrogen Cat#4392420 Assay ID: s32107 Transfected construct human Transfected construct (human) Non-targeting control Invitrogen Cat#4390846 Transfected construct human Transfected construct (human) siPORTamine Invitrogen Cat#AM4503 Transfected construct (human) Opti-MEM Invitrogen Cat#11058–021 Recombinant DNA reagent Ad5-U6-hLrrc8a-shRNAmCherry Vector biolabs shADV653 214592 Recombinant DNA reagent Ad5-U6-Scr-shRNAmCherry Vector biolabs 3086 Commercial assay or kit RNA isolation (PureLink RNA mini kit) Invitrogen 12183018A Software, algorithm GraphPad Prism8 La Jolla California USA, http://www.graphpad. com’ RRID:SCR_002798 Software, algorithm Fiji, ImageJ Schindelin et al., 2012 (PMID:22743772) RRID:SCR_002285 Other DAPI stain Thermofisher Cat#T3605 1:10,000

Techniques: Western Blot, Two Tailed Test