gfp atf6 plasmids  (TaKaRa)

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) A representative immunoblot of CD44 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. (B) Cell survival rate of shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment (n = 10). (C) Functional enrichment analyses of genes that were up- or downregulated (q value <0.05) by both shCD44–1 and shCD44–2 expression in IMR90-hTert cells. (D) Real-time qPCR data of representative UPR genes in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells (n = 3). Data were normalized to GAPDH . (E) A representative immunoblot of HSPA5 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. (F) Relative density of HSPA5/b-Actin analyzed by ImageJ (n = 3). (G) The boxplot shows the effects of CD44 overexpression on the expression levels of the indicated genes in IMR90 cells. The effects are shown for the genes that are upregulated by ATF6 activating molecule AA147 and the genes co-expressed with ATF6, ATF4, or XBP1 (retrieved from Enrichr). (H) Percentages of overlap between CD44 co-expressing genes (retrieved from COXPRESdb v7) and the indicated genes. (I) Relative cell survival rate of shCD44–1 and shCD44–2 IMR90-hTert cells compared with shLuc IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment. Vehicle (DMSO) or 10 μM HA15 was added to the medium during tunicamycin treatment (n = 10). All immunoblots were repeated once with similar results. Error bars are presented as mean ± SD values. *p < 0.05; one-way ANOVA with post hoc Dunnett’s test for (B and F), two-way ANOVA with post hoc Dunnett’s test for (D), Wilcoxon test for (G), Fisher’s exact test for (H), and two-tailed t test with Bonferroni-Dunn correction for (I).

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Western Blot, Functional Assay, Expressing, Over Expression, Two Tailed Test

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) Representative immunoblots of IRE1, PERK, and ATF6 in IMR90 cells 3 days after transfection with siLuc, siIRE1, siPERK, or siATF6. The experiment was repeated once with similar results. (B and D) Relative cell survival rate of shCD44–1 and shCD44–2 IMR90-hTert cells compared with shLuc IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment. (B) Cells were transfected with siLuc, siIRE1, siPERK, or siATF6 2 days before starting tunicamycin treatment (n = 10). (D) Vehicle (DMSO), 50 μM 4μ8C, 20 nM GSK2606414, 10 μM PF429242, 5 nM SCH772984, or 20 μM SB202190 was added to the medium during tunicamycin treatment. (C) Relative expression levels of representative UPR genes in IMR90 cells 24 h after 0.5 μg/mL tunicamycin treatment. Vehicle (DMSO), 50 μM 4μ8C, 20 nM GSK2606414, or 10 μM PF429242 was supplemented to the medium during tunicamycin treatment (n = 3). Expression levels were measured by real-time qPCR and normalized to GAPDH . Error bars are presented as mean ± SD values. *p < 0.05; two-way ANOVA with post hoc Dunnett’s test.

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Western Blot, Transfection, Expressing

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) Protein-protein interactions among the 41 CD44-associated proteins plus IRE1, PERK, and ATF6 were mapped using the STRING software. Only highest confidence interactions (interaction score ≥0.9) that are experimentally determined (purple line) or stored in curated databases (blue line) were included in the analysis. IRE1, PERK, and ATF6 are shown as red nodes and the 16 CD44-associated ER proteins are shown as blue nodes. CD44-associated proteins are defined here as proteins that are detected by cross-linked immunoprecipitation-mass spectrometry of IMR90 cells using anti-CD44 antibody with at least 10-fold greater abundance than in control experiment using normal rabbit IgG antibody. (B) Representative immunoblots of CALR, HSP47, and CD44 in whole cell lysate and in CD44-immunoprecipitates prepared from IMR90 cells. The experiment was repeated once with similar results. (C) Representative confocal images of IMR90 cells co-stained with antibodies against CD44 and organelle markers (mitochondrial marker mtTFA and ER markers ERp57, HSP47, and RPN2). (D) The dot plot shows Pearson correlation coefficients between immunofluorescence signals of CD44 and organelle markers. Each dot represents a single cell (n = 30). Error bars are presented as mean ± SD values. Scale bars, 10 μm.

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Software, Immunoprecipitation, Mass Spectrometry, Western Blot, Staining, Marker, Immunofluorescence

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) The heatmap shows relative abundances of ER-associated proteins detected by mass spectrometry of ER fractions isolated from shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Cells were collected 24 h after starting 0.5 μg/mL tunicamycin treatment. ER-associated proteins were defined here as the proteins whose ER localization have been confirmed by the Human Protein Atlas or associated with the GO term “endoplasmic reticulum lumen.” (B) Representative immunoblots of COL6A3, SEC62, and HSPA5 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Whole cell lysates and culture supernatants were collected 24 h after starting 0.5 μg/mL tunicamycin treatment. (C) Representative immunoblots of ATF6, IRE1, PERK, CALR, EGFR, and Vinculin in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Cells were lysed in buffer containing either 1% Triton or 2% SDS. (D) A representative immunoblot of ATF6 in U2OS cells and in ATF6 KO U2OS cells infected with empty vector or vector expressing wild-type ATF6, ATF6 Y392C, or ATF6 Y567N. (E) Cell survival rate of ATF6 KO U2OS cells after 5 days of 2 μg/mL tunicamycin treatment (n = 8). Cells were infected with vector expressing wild-type ATF6, ATF6 Y392C, or ATF6 Y567N and selected with 100 μg/mL hygromycin prior to the experiment. (F) Relative expression levels of HSP90B1 and HSP5A in CD44 KO U2OS cells (n = 3). Cells were infected with empty vector or vector encoding CD44-ectodomain-KDEL 3 days before sample collection. Data were normalized to GAPDH . (G) Cell survival rate of CD44 KO U2OS cells after 5 days of 2 μg/mL tunicamycin treatment. Cells were infected with empty vector or vector encoding CD44-ectodomain-KDEL 3 days before starting tunicamycin treatment (n = 8). All immunoblots were repeated at least once with similar results. Error bars are presented as mean ± SD values. *p < 0.05; two-way ANOVA with post hoc Dunnett’s test (E) and two-tailed t test with (F) or without (G) Bonferroni-Dunn correction.

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Mass Spectrometry, Isolation, Western Blot, Infection, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Recombinant, Transfection, Protease Inhibitor, CyQUANT Assay, Proliferation Assay, Expressing, Mass Spectrometry, Software