Journal: eLife

Article Title: Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

doi: 10.7554/eLife.47864

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pDONR-P2R-P3 (plasmid) , Invitrogen , , Gateway entry vector for tags/genes.

Techniques: Modification, Mutagenesis, Variant Assay, Recombinant, Plasmid Preparation, Activation Assay, Cloning, Sequencing, Staining, Software

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A) Experimental outline of HF or vehicle control DMSO treatment and DENV-2 envelope domain III (DENVrEDIII) protein immunization. (B) Proliferation analysis by 3H thymidine incorporation in antigen specific T-cells from splenocytes of mice 28 days after immunization that were restimulated with DENVrEDIII protein. Data are means ± SEM of 12 mice per treatment group from 2 independent experiments.(C to J) Flow cytometry analysis of DENVrEDIII-specific CD8+(C to F) and CD4+(G to J) T-cell responses in blood (D and H), spleen (E and I) and lymph node (F and J) after treatment and immunization, as indicated. FACS plots (C and G) are representative of 2 independent experiments. Quantification of the percentage of IFN-γ producing T-cells (D to F, and H to J) are means ± SEM of 12 mice per treatment group from 2 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two tailed unpaired student t test (B) and Mann-Whitney U test (D, E, F, H, I and J).

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Control, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A) Flow cytometry analysis of antigen-specific CD8+IFN-γ+ andCD4+IFN-γ+ T-cell kinetics in PBMCs of mice treated with DMSO or HF and immunized with DENV-2 envelope domain III after 14 and 28 days. Data are means ± SEM of 12 mice/group from 2 independent experiments. (B to D) Flow cytometry analysis of DENVrEDIII-specific polyfunctional CD8+ (B) and CD4+(C) T-cells in blood, spleen and lymph node of immunized mice. The frequency of double cytokine (IFN-γ and IL-2) producing cells with means (bar) ± SEM (B and C) and pie charts of the frequency of all cytokine producing T-cells (D) are from 12 mice/group from 2 independent experiments.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001by 2-way ANOVA with Bonferroni post-hoc between DENVrEDIII+DMSO and DENVrEDIII+HF immunized groups(A), Mann-Whitney U test(B and C)

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: ELISA analysis of the amounts of IFN-γ, IL-12p40 and TNF-α produced bysplenocytes and lymph node cells from immunized mice after re-stimulation in vitro with DENVrEDIII for 72hr. Spleen and lymph nodes were collected from mice at 28th day post-secondary immunization. Data are means ± SEM from 12 mice per treatment group from 2 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two tailed unpaired student t test.

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Produced, In Vitro, Two Tailed Test

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A) ELISA analysis of DENVrEDIII specific total IgG, IgG2a, IgG2b, and IgG1 in the serum of mice14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEM of 12 mice/group from 2 independent experiments. (B and C) BIAcore SPR analysis of DENVrEDIII protein binding by pooled serum samples from mice preconditioned with HF or DMSO 28 days after immunization. Sensogram trace of the DENVrEDIII-specific antibody binding affinity (B) and correlation of the Maximal Response Unit (RUmax) with the dissociation constant (C, upper) are representative of 2 independent experiments. Quantified avidity scores(C, lower) are means ± SEM from 2 independent experiments on pooled serum samples from 10 mice/group assayed in triplicate.(D) DENV-2 virus neutralization assay on serum samples collected from mice 28 days after immunization. The 50% focus reduction neutralization titre (FRNT50) data are means ± SEM of 10 mice from 2 independent experiments assayed in triplicate.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, by one-way ANOVA with Bonferroni post-hoc test (A), two tailed unpaired student t test(C) and Mann-Whitney U test (D).

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Protein Binding, Binding Assay, Virus, Neutralization, Two Tailed Test, MANN-WHITNEY

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A)ELISA of total IgG specific for all four DENV serotypes in the serum of mice immunized with a tetravalent combination of DENVrEDIII protein 14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEMof10 mice/group from 2 independent experiments. (B)DENV serotype neutralization assay on serum from tDENVrEDIII immunized mice. The 50% focus reduction neutralization titre (FRNT50) data are means ± SEM of 10 mice from 2 independent experiments. *P<0.05, **P<0.01 by one-way ANOVA with Bonferroni post-hoc test (A) and Mann-Whitney U test (B).

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, MANN-WHITNEY

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A and B) Confocal microscopy imaging of the GL-7+(red), B220+(green) IgG+(blue) GC B-cells in lymph node sections from mice treated with HF or DMSO and immunized with DENVrEDIII protein. Images (A) are representative of 2 independent experiments. Quantified data (B) are means ± SEM of 8 mice/condition from all experiments. (C) Flow cytometry analysis of lymph node GC-B cells frequency in DENVrEDIII immunized mice at the indicated time-points. Data are means ± SEM of 8 mice per group at each time-point from 2 independent experiments.(D) ELISA analysis of IL-10 production by splenocytes and lymph node cells from mice 28 days after secondary immunization that were restimulated with DENVrEDIII. Data are means ± SEM of 10 mice/group from 2 independent experiments. (E) qRT-PCR analysis of the indicated gene expression in lymph node cells restimulated with DENVrEDIII for 24hr. Heatmap of statistically significant changes are from the analysis of 10 biological replicates from 2 independent experiments. *P<0.05, **P< 0.01, ***P<0.001, ****P<0.0001by two tailed unpaired student t test (B and D) and 2-way ANOVA with Bonferroni Post-hoc test (C).

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Confocal Microscopy, Imaging, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: NIT-1 cells (10 7 ) transduced with the mouse GECKO A CRISPR lentiviral library (MOI=0.3) and selected with puromycin were implanted subcutaneously (SubQ) into NOD. scid mice, with or without intravenous injection of 10 7 splenocytes from diabetic NOD mice. After 8 weeks, NIT-1 grafts were retrieved from recipients with (autoimmune) and without (non-autoimmune) splenocyte co-injection. Next-generation sequencing of gRNAs present in surviving grafts identified Rnls gRNA (MGLibA_46009, 5’-CTACTCCTCTCGCTATGCTC-3’) as one of only 11 gRNAs detected at high frequency in mice with beta cell autoimmunity.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Transduction, CRISPR, Injection, Next-Generation Sequencing

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a: T7 endonuclease I assay. Genomic DNA from NIT-1 wild-type (WT) and Rnls mut cells was tested for CRISPR-Cas9 gene editing events. Cleavage at heteroduplex mismatch sites by T7 endonuclease I digestion was analyzed by agarose gel electrophoresis. DNA from Rnls mut cells segregated into multiple digested fragments, indicating efficient mutation of the targeted region in the Rnls gene. b: Genomic DNA from Rnls mut cells was sequenced to identify individual mutations. The Rnls gRNA targeting site is labelled in red. The frequency of the wild-type allele and of the most abundant mutations and their predicted consequence (frameshift / in-frame deletion) are shown. These frequencies indicate that 75% of the cells are predicted to carry at least one deleterious mutant allele. c: Islets (≈1700) were purified from 8-week old CD1 mice, dispersed and transduced with lentivirus encoding a non-targeting (NT) or Rnls -targeting gRNA together with the Cas9 endonuclease driven by the rat insulin promoter. 72 h later, islets were stimulated sequentially with 2.8 mM glucose, 16.8 mM glucose and finally 30 mM KCl to induce insulin secretion. Islet genomic DNA was quantified for normalization of ELISA insulin measurements to DNA content. n=5 technical replicates per condition and genotype. Data show mean ± SEM. Note that islet dispersion necessary for lentiviral transduction decreased the overall responsiveness of purified islets compared to intact islets. Insulin secretion by Rnls mutant islet cells was not significantly different from that of control (NT) islets. d : Growth curves for NIT-1 WT, control (NT gRNA) and Rnls mut cells seeded in 96-well plates at 50,000 cells/well over one week. Culture media were refreshed in every 2 days. Cell growth was measured on days 0, 3 and 7 using the CellTiter-Glo luminescence Cell Viability Assay (Promega). Growth rates were not significantly different as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. n=3 technical replicates per genotype. Data show mean ± SEM.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: T7EI Assay, CRISPR, Agarose Gel Electrophoresis, Mutagenesis, Purification, Transduction, Enzyme-linked Immunosorbent Assay, Dispersion, Control, Viability Assay

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a: Experimental approach used to test NIT-1 beta cell survival after transplantation and induction of autoimmunity. Control and Rnls mut NIT-1 cells (10 7 ) carrying a luciferase reporter were implanted on opposite flanks of NOD. scid mice. Autoimmunity was induced by injection of 10 7 splenocytes from diabetic (DM) NOD mice. b: Representative images of graft luminescence at days 0, 3, 7, 14 and 18 post-transplantation. c,d: Relative luminescence of paired Rnls mut and control grafts over time ( c ) and at day 18 ( d ), normalized to the ratio on day 0. * P =0.0357 at days 14 and 18 post-transplantation. Data show mean ± SEM of n=5 (+splenocytes) and n=3 (-splenocytes) mice. e: Experimental approach used to test NIT-1 cell survival transplanted into diabetic NOD mice. Control (NT) and Rnls mut NIT-1 cells (10 7 ) were implanted on opposing flanks of overtly diabetic NOD mice. f: Representative images of graft luminescence at day 0, 5, 8, 10, 14 and 18 post-transplantation. g: Proportion of remaining luminescence relative to day 0 (100%), * P =0.012. h: Relative luminescence of paired Rnls mut and control grafts. Data show mean ± SEM of n=5 mice. i: Experimental approach used to test autoimmune killing of primary islet beta cells. NOD. scid islet cells transduced with lentivirus encoding a non-targeting (NT) control or Rnls- targeting gRNA and RIP-driven Cas9 endonuclease were transplanted under the left and right kidney capsule, respectively, of the same NOD. scid recipients. Autoimmunity was induced as in ( a ). j: Representative images of transplanted islets on the explanted kidney at day 39. k: Quantification of insulin mRNA relative to glucagon ( Gcg ) mRNA in paired grafts from non-autoimmune (- splenocytes, n=5) and autoimmune (+ splenocytes, n=6) mice at day 39, * P =0.0312. l: Relative insulin expression in paired Rnls mut and control (NT) grafts, * P =0.0173. Data represent mean ± SEM, calculated by two-sided Mann-Whitney test ( c - d , g - h , l ), and two-sided Wilcoxon signed-rank test ( k ). All data are representative of two or more independent experiments.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Transplantation Assay, Control, Luciferase, Injection, Transduction, Expressing, MANN-WHITNEY

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: Control and Rnls mut NIT-1 cells (10 7 ) carrying a luciferase reporter were implanted on opposing flanks of NOD. scid ( a ) or C57BL/6 mice ( b , c ). a: Graft bioluminescence was measured on days 0 and 57 after transplantation of NIT-1 cells together with diabetogenic NOD splenocytes (as in ). b , c : Graft bioluminescence was measured on days 0, 4 and 7 after transplantation. Representative bioluminescence images ( b ) and relative luminescence of grafts over time ( c ) are shown (n=3). Data represent mean ± SEM. Both control and mutant grafts were destroyed by allo-rejection within a week.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Control, Luciferase, Transplantation Assay, Mutagenesis

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a-d: Representative flow cytometry data ( a , c ) and summary data ( b , d ) for MHC-I ( a , b , MFI: mean fluorescent intensity) and MHC-II ( c , d , expressed as % MHC-II + cells) expression in control and Rnls mut cells. Data are representative of three independent experiments. e,f: BDC2.5-TCR transgenic CD4 + T cells were co-cultured with NIT-1 cells and irradiated splenocytes from NOD. scid mice. IFN-γ expression in CD4 + T cells was measured at 24 h by flow cytometry. Representative ( e ) and combined data ( f ) from technical triplicates is shown. Data are representative of five independent experiments. g,h: ELISPOT measurement for the activation of polyclonal CD8 + T cells from a diabetic NOD mouse following stimulation with control or Rnls mut NIT-1 cells. Wells without NIT-1 cells or with PMA and ionomycin were used as negative and positive controls, respectively. Data are representative of three independent experiments. Data were compared by one-way ANOVA with Tukey’s multiple comparison test. ns, not significant, *** P =0.0001. All data show mean ± SEM.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Flow Cytometry, Expressing, Control, Transgenic Assay, Cell Culture, Irradiation, Enzyme-linked Immunospot, Activation Assay, Comparison

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a,b: Cell viability measurement 24 h after thapsigargin (TG) ( a ), tunicamycin (TC) ( b ) and cytokine (IL-1β and IFN-γ)( c ) treatment. Data show mean ± SEM of n=2–4 technical replicates per condition and are representative of 2–3 independent experiments. *** P < 0.0001, calculated by two-way ANOVA with Sidak’s multiple comparisons test. d-f: Measurement of the UPR in response to TG challenge. ER stress pathway protein phosphorylation (PERK, eIF2a and IRE1a), expression (ATF4, Txnip, NRF2) and cleavage (ATF6, Caspase3) ( d ), Xbp1 splicing ( e, *** P < 0.0001, ### P =0.0005) and Chop and Txnip mRNA levels ( f, *** P < 0.0001, # P =0.0268, ### P =0.0008) were measured in control and Rnls mut NIT-1 cells treated with or without 1 μM TG for 5 h. Data show mean ± SEM, n=3 per group and are representative of two independent experiments, calculated by one-way ANOVA with Dunnett’s multiple comparisons test. * Control vs. Rnls mut cells in non-treatment group; # Control vs. Rnls mut cells in TG-treatment group ( d and e ).

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Phospho-proteomics, Expressing, Control

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a: Viability of Rnls mut and control NIT-1 cells at 6 h and 24 h after treatment with 40 mM streptozotocin (STZ) (n=3 technical replicates). b: NIT-1 cell viability 48 h after mitomycin C (MMC) at the indicated concentration (n=3 technical replicates). *** P < 0.0001, calculated by two-way ANOVA with Sidak’s multiple comparisons test. c-f: Rnls knockout NIT-1 cell lines were generated by deleting either exons 2–4 or exon 5. Deletion efficiency was confirmed by qPCR of genomic DNA. Rnls ΔEx2/4 cells showed ~60% deletion of exons 2–4 genomic DNA qPCR ( c ) while Rnls ΔEx5 cells showed ~87% deletion of exon 5 ( d ). Cell viability of Rnls deficient cells was measured 72 h after thapsigargin (TG, e ) and tunicamycin (TC, f ) treatment. *** P < 0.0001, calculated by unpaired t-test ( c,d ) and two-way ANOVA with Sidak’s multiple comparisons test ( e,f ). g,h: Overexpression of Rnls in WT NIT-1 cells increased sensitivity to low dose-TG-induced killing ( g ). n=4 technical replicates per group. *** P < 0.0001, calculated by two-way ANOVA with Sidak’s multiple comparisons test. CRISPR-immune Rnls (CiRnls) expressed in Rnls mut cells restored sensitivity to TG-induced killing ( h ). n=4 technical replicates per group. *** P < 0.001, # P = 0.0138, ### P = 0.0002, 0.0005 for 0.05 and 0.25 TG(μM) respectively, calculated by two-way ANOVA with Sidak’s multiple comparisons test. *Comparison of control vs. Rnls mut cells; #comparison of Rnls mut vs. Rnls mut + CiRnls cells. All data represent mean ± SEM.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Control, Concentration Assay, Knock-Out, Generated, Over Expression, CRISPR, Comparison

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a-c: Control (WT) and Rnls overexpressing (Rnls OE ) NIT-1 cells carrying a luciferase reporter were implanted on opposing flanks of NOD. scid mice. Some graft recipients were also injected intravenously with splenocytes from diabetic NOD mice (DM NOD splenocytes). Graft bioluminescence was imaged on days 0, 2, 3 and 7 ( a ). The relative luminescence of Rnls OE and control grafts over time, normalized to day 0, is shown in ( b ). Data for all mice analyzed on day 3 is shown in ( c ). Rnls OE graft were more sensitive to autoimmune killing as evidenced by more rapid loss of luminescence. By day 7, both control and Rnls OE grafts were killed to ~90% (data not shown), resulting in a similar relative luminescence level. n=6 mice (each with two grafts). Data represent mean ± SEM, ** P < 0.0022, calculated by two-sided Mann-Whitney test. d,e: Rnls mut NIT-1 cells and Rnls mut cells expressing the CRISPR-immune Rnls transgene (CiRnls), all carrying a luciferase reporter, were implanted on opposing flanks of NOD. scid mice. Graft recipients were also injected intravenously with splenocytes from diabetic (DM) NOD mice. Graft bioluminescence was imaged on days 0, 2, 3 and 5 post-injection ( d ). Relative luminescence of paired grafts over time normalized to day 0 is shown in ( e ). n=5 mice. Data represent mean ± SEM.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Control, Luciferase, Injection, MANN-WHITNEY, Expressing, CRISPR

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a: Quantification of Western blot data shown in . Images were obtained and quantified using a C-DiGit scanner and the Image Studio software (LI-COR Biosciences). n=3 per group. Data show mean ± SEM, *# P < 0.05, **## P < 0.01, ***### P < 0.001, calculated by one-way ANOVA with Dunnett’s multiple comparisons test. *Comparison to control cells without TG treatment; #comparison to control cells with 5-hour TG treatment. b: Control (Ctrl) and Rnls mut NIT-1 cells were cultured overnight with or without hydrogen peroxide (H 2 O 2 ) at the indicated concentrations. Cell viability was assessed using the CellTiter-Glo luminescence Cell Viability Assay. Data show mean ± SEM of triplicate cultures and are representative of three independent experiments. **** P<0.0001, calculated by two-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Western Blot, Software, Comparison, Control, Cell Culture, Viability Assay

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a: Structural model of Pargyline in complex with human RNLS (hRNLS). The protein is shown in cartoon and surface representation. Pargyline (green) and co-factor FAD (orange) are shown as sticks. b: Human recombinant RNLS protein denaturation profile in the presence and absence of pargyline (PG) by a SYPRO orange protein-dye-based thermal shift assay. c: RNLS melting temperature (Tm) change in response to pargyline. Data were fit to a variable slope four-parameter sigmoid curve. n=3 technical replicates per condition, representative of two independent experiments. d,e: Apoptotic response measured by caspase-3 activation in NIT-1 cells ( d , * P = 0.0112, # P = 0.0223) and NOD. scid islet cells ( e , * P = 0.0359 and 0.0495 for +PG and +PG/TG group respectively, ** P = 0.0065, ### P = 0.0003) 5 hours after thapsigargin (TG) treatment in the presence or absence of PG. Data show mean ± SEM of n=3 per condition, calculated by one-way ANOVA with Dunnett’s multiple comparisons test. * Comparison to WT or islet; # comparison to WT+TG or +TG. f: Experimental approach used to test pargyline for the protection of NIT-1 cells transplanted into diabetic NOD mice. g: Representative images of graft luminescence at days 0, 3, 5, 7, 12 and 19 post- transplantation. h: Proportion of remaining graft luminescence relative to day 0 (100%). i: Blood glucose levels in the control and pargyline treated mice over time, * P = 0.0316, 0.0053, 0.0367, 0.0422, 0.0381 and 0.0357 at days 5, 7, 9, 14, 19 and 20. Data show mean ± SEM of n=5 mice per group and are representative of three similar experiments, calculated by two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Recombinant, Thermal Shift Assay, Activation Assay, Comparison, Transplantation Assay, Control

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a: RNLS dual-gRNA design for the generation of RNLS knockout (KO) human induced pluripotent stem cells (SC). b: Genotyping of SC clones. CRISPR targeted clones were genotyped by PCR that was repeated for confirmation for all mutant clones, and individual mutations were verified by sequencing. Clone 1 was used as RNLS KO in this study and carried a 112bp deletion on both alleles. c: Glucose stimulated insulin secretion by SC-beta cells differentiated from WT or RNLS KO isogenic SC clones. Data in c show mean insulin secretion from four independent SC-beta cell batches, each measured in triplicate, following stimulation with 2.8mM glucose, 20mM glucose, or 30mM potassium chloride (KCl). Data show mean ± SEM for n=4 technical replicates per condition and genotype.

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Knock-Out, Clone Assay, CRISPR, Mutagenesis, Sequencing

Journal: Nature metabolism

Article Title: Genome scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes

doi: 10.1038/s42255-020-0254-1

Figure Lengend Snippet: a: Schematic representation of the differentiation protocol used to generate SC-beta cells from isogenic wild-type (WT) and RNLS KO human iPSC. b,c: Representative flow cytometry analyses ( b ) and quantification ( c ) of the proportion of differentiated iPSC cells that co-expressed C-peptide and NKX6.1 as markers of beta cell identity. Data show mean ± SEM of n=6 (WT) and n=4 (RNLS KO). d,e: Representative flow cytometry analyses ( d ) and quantification ( e , * P = 0.028 and 0.0348 for RNLS KO and RNLS KO+TG group respectively, *** P < 0.0001, ### P < 0.0001) of SC-beta cell death following TG treatment, measured by the apoptosis dye apopxin within CD49a + beta cells . Data show mean ± SEM of n=4 per condition and genotype. * Comparison to WT; # comparison to WT+TG. f,g: Apoptosis in CD49a + iPSC- ( f ) and embryonic stem cell- (HUES8) ( g ) derived beta cells challenged with TG in the presence or absence of pargyline (PG), n=6 per condition and genotype. Data show mean ± SEM, calculated by two-tailed unpaired t-test ( c ) and one-way ANOVA with Dunnett’s multiple comparisons test ( e-g ).

Article Snippet: Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP.

Techniques: Flow Cytometry, Comparison, Derivative Assay, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus

doi: 10.1186/s13287-021-02324-7

Figure Lengend Snippet: Elevating CXCR7 expression enhanced the functional activity of EPCs. a , b CXCR7 expression in diabetic EPCs (diabetic group) and normal EPCs (normal group) ( n = 5). c , d Capacity of EPCs in different groups to adhere to HUVECs (bar = 100 μm; n = 5). e , f Repair capacity of EPCs in different groups (bar = 200 μm; n = 5) (* p < 0.05, compared with group diabetes; & p < 0.05, compared with group SiCXCR7; # p < 0.05, compared with group Lv-Ctrl)

Article Snippet: Recombinant lentivirus encoding CXCR7 was constructed using the pLVX-EGFP-3FLAG-Puro vector (Shanghai Sunbio Medical Biotechnology, Shanghai, China).

Techniques: Expressing, Functional Assay, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus

doi: 10.1186/s13287-021-02324-7

Figure Lengend Snippet: Upregulating CXCR7 expression accelerated reendothelialization and inhibited neointimal hyperplasia. a Representative images of Evans blue staining (bar = 500 μm; n = 5 per group). b Reendothelialization rate in different groups after 7 days. c Reendothelialization rate after 14 days. d I / M ratio of HE-stained EPCs 21 days after treatment (bar = 150 μm; n = 5 per group). e Neointimal area assessed by Masson trichrome staining 21 days after treatment (bar = 150 μm; n = 5 per group) (* p < 0.05; ** p < 0.001)

Article Snippet: Recombinant lentivirus encoding CXCR7 was constructed using the pLVX-EGFP-3FLAG-Puro vector (Shanghai Sunbio Medical Biotechnology, Shanghai, China).

Techniques: Expressing, Staining

Journal: Stem Cell Research & Therapy

Article Title: Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus

doi: 10.1186/s13287-021-02324-7

Figure Lengend Snippet: Upregulating CXCR7 expression activated the Akt-associated Keap-1/Nrf2 axis. a Levels of p-Akt, Keap-1, n-Nrf2, HO-1, and NQO-1. b Expression of HO-1 and NQO-1 after blockade of Nrf2 (* p < 0.05; ** p < 0.001)

Article Snippet: Recombinant lentivirus encoding CXCR7 was constructed using the pLVX-EGFP-3FLAG-Puro vector (Shanghai Sunbio Medical Biotechnology, Shanghai, China).

Techniques: Expressing

Journal: Stem Cell Research & Therapy

Article Title: Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus

doi: 10.1186/s13287-021-02324-7

Figure Lengend Snippet: Knockdown of Nrf2 attenuated the functional activity of CXCR7-EPCs. a Differences in adhesion capacity among all groups (bar = 100 μm; n = 5). b Differences in repair capacity among all groups (bar = 200 μm; n = 5) (* p < 0.05, compared with control group; & p < 0.05, compared with group CXCR7-EPC Nrf2-KD group)

Article Snippet: Recombinant lentivirus encoding CXCR7 was constructed using the pLVX-EGFP-3FLAG-Puro vector (Shanghai Sunbio Medical Biotechnology, Shanghai, China).

Techniques: Knockdown, Functional Assay, Activity Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus

doi: 10.1186/s13287-021-02324-7

Figure Lengend Snippet: Knockdown of Nrf2 impaired endothelial repair of CXCR7-EPCs in vivo. a Representative images of Evans blue staining after EPC transplantation (bar = 500 μm; n = 5 per group). b Reendothelialization rate in different groups after 7 days. c Reendothelialization rate in different groups after 14 days. d Representative images of HE and Masson trichrome staining (bar = 150 μm; n = 5 per group). e I / M ratio in different groups after EPC transplantation. f Neointimal area in different groups after EPC transplantation (* p < 0.05; ** p < 0.001)

Article Snippet: Recombinant lentivirus encoding CXCR7 was constructed using the pLVX-EGFP-3FLAG-Puro vector (Shanghai Sunbio Medical Biotechnology, Shanghai, China).

Techniques: Knockdown, In Vivo, Staining, Transplantation Assay

Journal: Stem Cell Research & Therapy

Article Title: Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus

doi: 10.1186/s13287-021-02324-7

Figure Lengend Snippet: The protective effect of the SDF-1/CXCR7 axis on diabetic EPCs. Diabetes decreased the expression of CXCR7 in diabetic EPCs and impaired the adhesion and repair capacities of EPCs in vitro and in vivo. However, elevating CXCR7 expression enhanced the functional activity of EPCs by activating the Akt-associated Keap-1/Nrf2 axis, counteracting the oxidative stress damage induced by ROS in DM

Article Snippet: Recombinant lentivirus encoding CXCR7 was constructed using the pLVX-EGFP-3FLAG-Puro vector (Shanghai Sunbio Medical Biotechnology, Shanghai, China).

Techniques: Expressing, In Vitro, In Vivo, Functional Assay, Activity Assay

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Schematic of the protocol for generation of tdTomato-CD63 + BV-2 cells and lentivirus-induced expression of tGFP-shRNA targeting Sepp1 , created with BioRender. (B) Transduced tdTomato-CD63 + tGFP-shRNA + BV-2 cells were sorted by FACS. (C) Representative images of double-positive (tdTomato + /tGFP + ) BV-2 cells transduced with tGFP-shRNA for downregulation of Sepp1 . Scale bar, 50 μm. (D) Workflow for purification of EVs from the conditioned medium (CM) of tdTomato-CD63 + BV-2 microglia upon stimulation with LPS and ATP; created with BioRender . (E–G) Size distribution and concentration of EVs secreted on CM of BV-2 cells were measured by nanoflow cytometry using a flow nanoanalyzer (NanoFCM); ** p < 0.01 and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). The assessment of tdTomato-CD63 + molecule loading on EVs was performed using the Nanoimager system (ONI). (H) Representative image of single tdTomato-CD63 + EVs. Scale bar, 2 μm. (I and J) Single-molecule fluorescence analysis of tdTomato-CD63 loading on EVs was performed at single-EV level, and the number of tdTomato-CD63 molecules present in single EVs was quantified using the CODI platform from ONI; one-way ANOVA with Holm-Šidák post hoc analysis ( n = 3 replicates per group). * p < 0.05, ** p < 0.01, and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 per group). Scale bar, 50 nm. Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Expressing, shRNA, Transduction, Purification, Concentration Assay, Cytometry, Fluorescence

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Principal-component analysis of transcriptomic profile of tdTomato-CD63 + BV-2 cells transduced with tGFP-shRNA targeting scramble and Sepp1 (three different clones) ( n = 6 replicates per group). (B) Heatmap displaying the Pearson correlation of scramble and Sepp1-shRNA replicates from RNA sequencing (RNA-seq). (C–E) Volcano plots comparing the DEGs of three Sepp1 -shRNA clones versus scramble. Dashed lines indicate a significant threshold using q < 0.05 (−log10 q- value > 1.3) on the y axis and log2 fold change > +0.5 or < −0.5 on the x axis. (F) Venn diagram comparison of up- and downregulated DEGs across different Sepp1 -shRNAs clones using the same criteria as in (D) and (E) ( q < 0.05 and log2 fold change > +0.5 or < −0.5). (G) Common Sepp1 -shRNA DEGs were subjected to Gene Ontology classification, and the top 5 significant ( q < 0.05) for each cellular component (CC), biological process (BP), and molecular function (MF) are displayed as a bar graph based on gene count enrichment.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Transduction, shRNA, Clone Assay, RNA Sequencing, Comparison

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Top 10 enriched cellular processes identified by KEGG pathway analysis of all common regulated DEGs by Sepp1 -shRNA in BV-2 microglia. (B) Heatmap showing the consistent regulation of the components of endocytosis and lysosome pathways from (A) by different Sepp1 -shRNAs in BV-2 microglia. Identified components related to endosome/exosome ESCRT machinery, the retromer complex, and lysosomal activity are highlighted in bold. (C–E) Bar graphs depicting normalized expression levels of highlighted genes from (B) among scramble and Sepp1 -shRNA groups in RNA-seq data. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). Bar graphs indicate mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: shRNA, Activity Assay, Expressing, RNA Sequencing

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Heatmap displaying the top common DEGs ( q < 0.05) regulated in response to Sepp1 downregulation. (B) Enriched pathways associated with the common DEGs in Sepp1 -shRNA were obtained using Metascape software. (C and D) Ingenuity Pathway Analysis (IPA) using all common DEGs regulated in different Sepp1 -shRNA groups predicted LXR/RXR pathway downregulation and identified a network interaction composed mainly of Ptger4 , Npc1 , Trem2 , and Cop1 in response to Sepp1 downregulation (D). (E) Analysis of normalized expression of Ptger4 , Npc1 , Trem2 , and Cop1 in RNA-seq data. * p < 0.05, ** p < 0.01, and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). Bar graphs indicate mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: shRNA, Software, Expressing, RNA Sequencing

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Schematic of the experimental design for Sepp1 silencing using siRNA in primary neurons and primary astrocytes. (B) Representative MAP2 and GFAP immunostaining images in primary neurons and astrocytes. Scale bar, 100 μm. (C) Bar graphs showing relative expression of Sepp1 mRNA in primary neurons and astrocytes treated with siRNA targeting the scramble control sequence or Sepp1 ( n = 6 replicates per group from 2 independent experiments). **** p < 0.0001 by two-tailed student t test. (D and E) Size distribution and concentration of EVs secreted in CM of primary neurons (D) and astrocytes (E) measured by nanoflow cytometry (NanoFCM) ( n = 6–8 replicates per group from 2 independent experiments). * p < 0.05 by two-tailed student t-test; ns, no significance. Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Immunostaining, Expressing, Control, Sequencing, Two Tailed Test, Concentration Assay, Cytometry

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Experimental design for microglia-specific EV labeling and Sepp1 silencing in APP NL-G-F mice. Six-month-old mice received injections into the hippocampus with a lentivirus for microglia-specific expression of mEm-CD9, followed by a lentivirus injection for scramble-shRNA:mCherry or Sepp1 -shRNA:mCherry expression 2 weeks later. (B) Representative images of mEm-CD9 + (green) microglia cells and cells expressing mCherry:shRNA (red) in the APP NL-G-F mouse dentate gyrus in the hippocampus brain region. Scale bar, 100 μm. (C) Confocal microscopy and Imaris 3D rendering of mouse hippocampal tissue. Shown are mEm-CD9 + (green) and P2ry12 + (red). Scale bar, 100 μm. (D) Percentage of the P2ry12 microglial marker overlapped with mEm-CD9 + cells in the hippocampus of APP NL–G-F mice injected with shRNA. (E) Super-resolution confocal z stack images of single microglia co-transduced with mEm-CD9 and shRNA:mCherry in the hippocampus of APP NL-G-F mice. 3D images of shRNA:mCherry (red) and Mac2 (magenta) surfaces overlapped with mEm-CD9 + (green) microglia as well as mEm-CD9 + particles surrounding microglia were generated using Imaris software. Scale bar, 4 μm. (F) Measured Mac2 fluorescence intensity in scramble- and Sepp1-shRNA-transduced microglia in APP NL-G-F mice ( n = 3–4 microglia per mouse, 3–4 mice per group). Two-tailed student t test; ns, no significance. (G) Representative high-magnification images of mEm-CD9 + voxels obtained in areas surrounding Mac2 + MGnD microglia as in (E). Scale bars: total EVs, 1 μm; small EVs, 0.2 μm. (H–K) In situ analysis of mEm-CD9 + voxels surrounding mEm-CD9 + /Mac2 + MGnD microglia. Shown are (H) the size frequency distribution of mEm-CD9 + particles and quantification of the number of mEm-CD9 + voxels per microglia in total EVs (I, total EVs <1,000 nm), small EVs (J, <150 nm), and large EVs (K, >150 nm) among scramble- and Sepp1 -shRNA:mCherry-transduced mEm-CD9 + /Mac2 + MGnD microglia. Two-tailed student t test, * p < 0.05 ( n = 3–4 microglia per mouse, 3–4 mice per group). Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Labeling, Expressing, Injection, shRNA, Confocal Microscopy, Marker, Transduction, Generated, Software, Fluorescence, Two Tailed Test, In Situ

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Virus, shRNA, Control, Recombinant, Staining, RNA Sequencing, Software, Saline, Imaging