Journal: Cellular and Molecular Immunology

Article Title: ADAM12 is a costimulatory molecule that determines Th1 cell fate and mediates tissue inflammation

doi: 10.1038/s41423-020-0486-8

Figure Lengend Snippet: ADAM12 acts as a costimulatory molecule, contributing to T-cell activation by amplifying TCR signaling. Human CD4 + T cells were purified from healthy donors and seeded on plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2 μg/ml) for 6 days, and anti-ADAM12 7G3 (10 μg/ml, full antibody or Fab), 7B8 (10 μg/ml, full antibody or Fab), and ADAM12-S (5 ng/ml) were added to the cultures separately. Cells were pulsed with BrdU for another 6 h. a Representative FACS dot plots of percentages of ADAM12 expression on untreated CD3 + CD4 + T cells. b DNA synthesis was measured on an ELISA reader; proliferation assay results shown as O.D. values. Data are mean ± SEM of five independent experiments. N = 9; one-way ANOVA with Bonferroni’s multiple comparisons test was used, * P < 0.05, ** P < 0.01. Student’s t test was used to show significant differences between anti-CD3 and anti-CD3 + anti-CD28, ϕ P < 0.05. Human CD4 + T cells were purified from healthy donors. CD4 + T cells were labeled with CFSE prior to being seeded on plate-bound anti-CD3 (5 μg/ml), and soluble anti-CD28 (1 μg/ml) and 7B8 (10 μg/ml) were added to the cultures for 4–5 days as indicated. c Representative FACS dot plots of CFSE gating. d Quantification of percentage of CFSE + cells. e Representative FACS dot plots of pZAP-70 staining as indicated. f Quantification of percentage of pZAP-70 + CD4 + T cells. g Representative FACS dot plot of CD69 staining as indicated. h Quantification of percentage of CD69 + CD4 + T cells. Graphs in d , f , and h are mean ± SEM from three independent experiments. N = 3–4 per group. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with post-Tukey’s test. ADAM12 +/+ and ADAM12 −/− spleens were taken from MOG 35–55 -induced EAE mice. Single-cell suspensions were treated with plate-bound anti-CD3 (5 μg/ml) and anti-ADAM12 (10 μg/ml) for 3 days prior to being stained with FACS antibodies. i FACS dot plots of the gating strategy; cells were gated on CD3 + CD4 + T cells. Representative FACS dot plots show percentages of Ki-67 and pZAP-70 expression on T cells under various conditions. j Percentage of Ki-67 + and k pZAP-70 + Ki-67 + cells. Data are mean ± SEM from three independent experiments. N = 3 was used. * P < 0.05, ** P < 0.01, *** P < 0.001 by two-way ANOVA and post-Tukey’s multiple comparisons test. Student’s t test was used to analyze CD3 treatment effects between ADAM12 +/+ and ADAM12 –/– T cells. φφφ P < 0.001. l DNA synthesis was measured on an ELISA reader; proliferation assay results are shown as O.D. values. Data are mean ± SEM from four independent experiments. N = 9. * P < 0.05 by one-way ANOVA with Bonferroni’s multiple comparisons test. Jurkat cells were transfected with wild-type (ADAM-L) and catalytically inactive (E351-A) full-length human ADAM12-L constructs. After 48 h, Jurkat T cells were stimulated overnight with ImmunoCult™ Human CD3/CD28 T-Cell Activator. m BrdU cell proliferation assay; relative values calculated as OD value divided by OD value in non-treated cells. Data are mean ± SEM from two independent experiments. N = 6. *** P < 0.001 by one-way ANOVA with post-Tukey’s test. n N-TIMP-2 (100 nM) and BB-94 (20 nM) were added to the cultures overnight prior to the cell proliferation assay. Data are mean ± SEM from two independent experiments. N = 6. *** P < 0.001 by two-way ANOVA and post-Tukey’s multiple comparisons test

Article Snippet: Cell proliferation was measured using the BrdU Cell Proliferation Assay Kit (K306–200, Biovision).

Techniques: Activation Assay, Purification, Expressing, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Labeling, Staining, Transfection, Construct, BrdU Cell Proliferation Assay

Journal: Cellular and Molecular Immunology

Article Title: ADAM12 is a costimulatory molecule that determines Th1 cell fate and mediates tissue inflammation

doi: 10.1038/s41423-020-0486-8

Figure Lengend Snippet: ADAM12 regulates the severity of neurological symptoms of EAE. Mean clinical EAE score in ADAM12 +/+ , ADAM12 +/− , and ADAM12 –/– mice; N = 24–32 mice per group. * P < 0.05 by Kruskal–Wallis with post-Dunn’s multiple comparisons test between ADAM12 –/– and ADAM12 +/+ . Mann–Whitney test shows a significant difference on day 12 ( ADAM12 –/– vs ADAM12 +/– ), ν P < 0.05, and on days 21, 23, and 25, respectively ( ADAM12 –/– vs ADAM12 +/+ ), φ P < 0.05, φφ P < 0.01. b Mean max score. c Cumulative score and d area under the curve (AUC) were calculated separately in ADAM12 +/+ , ADAM12 +/– , and ADAM12 –/– groups; data are mean ± SEM from two independent experiments. N = 24–33 mice per group. Kruskal–Wallis with post-Dunn’s multiple comparisons test shows a significant difference in mean max score between ADAM12 –/– and ADAM12 +/+ mice, * P < 0.05. Mann–Whitney test shows a significant difference in cumulative score and AUC between ADAM12 –/– and ADAM12 +/+ mice, * P < 0.05. e Representative H&E images of spinal cords from mice with EAE; scale bars = 500 μm. f Cell counts after purification of CNS-infiltrating cells; data are mean ± SEM from three independent experiments. N = 6–7. * P < 0.05 by Student’s t test. Manual quantification of the number of CNS-infiltrating cells from histology slides g inside and h outside of vessels. N = 32–34 tissue sections per group. Data are mean. * P < 0.05 by Student’s t test. i FACS analysis of percentage of CD4 + T cells from purified CNS-infiltrating cells. Data are min to max from five independent experiments. N = 4–5. * P < 0.05 by Mann–Whitney test. j Immunofluorescence staining of IFNγ + TCRα + T cells among CNS-infiltrating cells: TCRα (green), IFNγ (red), and DAPI (blue); scale bars = 10 μm. k Quantification of the number of IFN-γ + CD3 + among CNS-infiltrating cells in ADAM12 +/+ and ADAM12 −/− EAE mice; data are mean ± SEM from three independent experiments. N = 6 mice per group. * P < 0.05 by Student’s t test. l Immunofluorescence staining of IL-17 + TCRα + T cells among CNS-infiltrating cells: TCRα + (green), IL-17 (red), and DAPI (blue); scale bars = 10 μm. m Quantification of the number of IL-17 + TCRα + T cells among CNS-infiltrating cells in ADAM12 +/+ and ADAM12 −/− EAE mice; data are mean ± SEM from three independent different experiments. N = 6 mice per group. Spleens were taken from ADAM12 +/+ and ADAM12 −/− EAE mice, single-cell suspensions were established and treated with MOG 35–55 (/ml) for 48 hours, cells were pulsed with BrdU for another 6 h, and DNA synthesis was measured on an ELISA reader. n DNA synthesis (O.D. value) of T-cell proliferation in MOG 35–55 -treated splenocytes. Data are mean ± SEM from three independent experiments. N = 6. * P < 0.05 by Mann–Whitney test. Cell culture supernatants were collected, and o IFN-γ and p IL-17 production in splenocyte cell cultures was measured by ELISA. ConA (Concanavalin A, 5 μg/ml) was used as a positive control. Data are mean ± SEM from three independent experiments. N = 12–15. ** P < 0.01 by Mann–Whitney test for comparison between ADAM12 +/+ versus ADAM12 −/− . q Mean clinical EAE score of C57BL/6 mice treated with MOG 35–55 and anti-ADAM12 Fab (7B8 + 7G3). Purified monoclonal lgG was used as an isotype control. N = 10 mice per group. ** P < 0.01 by Kruskal–Wallis test, showing significant difference between anti-ADAM12 Fab-treated and PBS groups. ϕϕ P < 0.01 by Mann–Whitney test, showing significant difference between anti-ADAM12 Fab-treated and isotype control groups. Mann–Whitney test shows a significant difference on days 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, and 25 (7B8 + 7G3 vs PBS) : φ P < 0.05, φφ P < 0.01 and on days 20, 21, 22, and 23 (7B8 + 7G3 vs isotype): ν P < 0.05, νν P < 0.01. r Cumulative score of three groups. Data are mean ± SEM; N = 10 mice per group. * P < 0.05 by Kruskal–Wallis test, showing significant difference between antibody-treated and PBS groups. ϕ P < 0.05 by Mann–Whitney test, showing significant difference between antibody-treated and isotype control groups. s Area under curve (AUC) values were calculated separately. Graphs are mean ± SEM; N = 10 mice per group. ** P < 0.01, *** P < 0.001 by one-way ANOVA with post-Tukey’s multiple comparison. t Spleens were taken from EAE mice, single-cell suspensions were established and treated with MOG 35–55 (50 μg/ml) for 48 h, and cell culture supernatants were collected for ELISA. Th1, Th2, and Th17 cytokines were measured by ELISA in the supernatants, pooled for each group. Data are mean from ten splenocyte cultures. u IFNγ production by MOG 35–55 -treated splenocyte cultures. Graphs are mean ± SEM. N = 6–9. ** P < 0.01 by Kruskal–Wallis test

Article Snippet: Cell proliferation was measured using the BrdU Cell Proliferation Assay Kit (K306–200, Biovision).

Techniques: MANN-WHITNEY, Purification, Immunofluorescence, Staining, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Cell Culture, Positive Control

Journal: Communications Biology

Article Title: Bob1 maintains T follicular helper cells for long-term humoral immunity

doi: 10.1038/s42003-024-05827-0

Figure Lengend Snippet: a – d Adoptive cell transfer experiments of CD4 + Tcm and Tem cells. a Experimental protocol for immunized cell transfer. OVA was administered intraperitoneally with CFA on day 0 to CD45.2 + FoxP3 GFP/DTR CD4 Cre/+ Bob1 fl/fl mice and control CD45.2 + FoxP3 GFP/DTR CD4 +/+ Bob1 fl/fl mice. Tcm cells (GFP - B220 - CD4 + CD44 + CD62L + ) or Tem cells (GFP - B220 - CD4 + CD44 + CD62L - ) from the spleens of immunized mice were sorted on day 42 and transferred to CD45.1 + TCRβ −/− mice via tail veins using the sorting strategy shown in Supplementary Fig. . The next day, mice were immunized intraperitoneally with OVA in IFA, and then the sera and spleen cells were analyzed 7 days later. b Representative flow cytometric profiles and graphs of CD45.2 + Tfh cells (CD4 + CD45.2 + CXCR5 + PD-1 + ) in CD45.1 + TCRβ −/− hosts transferred with CD45.2 + Tcm or Tem cells. The % of CD45.2 + Tfh cells among the total CD4 + CD45.2 + T cells are shown. * P = 0.0159 (CD4 +/+ Bob1 fl/fl Tcm cells vs. CD4 Cre/+ Bob1 fl/fl Tcm cells) and † P = 0.0159 (CD4 +/+ Bob1 fl/fl Tcm cells vs. CD4 +/+ Bob1 fl/fl Tem cells). c Representative flow cytometric profiles and graphs of CD45.2 + ICOS + Tfh cells in CD45.1 + TCRβ −/− hosts transferred with CD45.2 + Tcm cells. The % of CD45.2 + ICOS + Tfh cells among the total CD45.2 + Tfh cells are shown. d Serum levels of OVA-specific IgG1 antibodies in CD45.1 + TCRβ −/− hosts transferred with CD45.2 + Tcm cells as assessed by ELISA. * P = 0.0159. e – g Histological distribution of Tfh cells originated from CD4 + Tcm cells. e Experimental protocol for immunized cell transfer. OVA was administered intraperitoneally with CFA on day 0 to CD45.1 + OT-II + FoxP3 GFP/DTR CD4 Cre/+ Bob1 fl/fl mice and control CD45.1 + OT-II + FoxP3 GFP/DTR CD4 +/+ Bob1 fl/fl mice. CD45.1 + OT-II + CD4 + Tcm cells were sorted on day 42 and then intravenously transferred into CD45.2 + wild-type mice. The next day, mice were immunized intraperitoneally with OVA emulsified in IFA. Seven days later, spleens were examined by immunostaining with a laser confocal microscope. f Representative confocal images of spleen sections stained for CD45.1 (green), PNA (red), and DAPI (blue). Scale bar: 50 μm. g Graphs show the fluorescence intensity of transferred cells (CD45.1 + OT-II + ) localized within the PNA + GC areas of CD45.2 + wild-type hosts. Imaging data were analyzed by ZEN software. ** P = 0.0020. h , i Dual DNA labeling experiment. h Experimental protocol for immunization in FoxP3 GFP/DTR CD4 Cre/+ Bob1 fl/fl mice and control FoxP3 GFP/DTR CD4 +/+ Bob1 fl/fl mice. NP 16 -OVA was injected intraperitoneally with CFA on day 0 and with PBS on day 30. BrdU was administered intraperitoneally on days −1, 1, 3, 5, and 7. On day 37, EdU was administered through the tail vein 3 h before analysis. i Representative flow cytometric profiles and graphs of BrdU + EdU + Tfh cells (FoxP3 - CD3 + CD4 + CXCR5 + PD-1 + ) derived from spleens. The % of BrdU + EdU + Tfh cells among the total Tfh cells are shown. ** P = 0.0015. Data represent the mean ± SD in ( b , c , d , g , i ) of 4–11 mice per group. Statistical significance was analyzed by the Mann-Whitney U -test for all groups: * P < 0.05, ** P < 0.01. Data in ( b – d ) are shown from FoxP3 GFP/DTR CD4 +/+ Bob1 fl/fl mice (open triangle) and FoxP3 GFP/DTR CD4 Cre/+ Bob1 fl/fl mice (closed triangle), and data in ( g ) are shown from CD45.1 + OT-II + FoxP3 GFP/DTR CD4 +/+ Bob1 fl/fl mice (open triangle) and CD45.1 + OT-II + FoxP3 GFP/DTR CD4 Cre/+ Bob1 fl/fl mice (closed triangle). Data in ( i ) are shown from FoxP3 GFP/DTR CD4 +/+ Bob1 fl/fl mice (open triangle) and FoxP3 GFP/DTR CD4 Cre/+ Bob1 fl/fl mice (closed triangle). Similar results were obtained across two to three independent experiments.

Article Snippet: To detect incorporated BrdU and EdU in DNA, cells were analyzed with a BrdU assay kit for flow cytometry using an APC-conjugated anti-BrdU antibody (BD Biosciences) and a Click-iT EdU Pacific Blue flow cytometry assay kit utilizing click chemistry of azide-alkyne reactions (Thermo Fisher Scientific).

Techniques: Enzyme-linked Immunosorbent Assay, Immunostaining, Microscopy, Staining, Fluorescence, Imaging, Software, DNA Labeling, Injection, Derivative Assay, MANN-WHITNEY

Journal: PeerJ

Article Title: Identification of CDT1 as a prognostic marker in human lung adenocarcinoma using bioinformatics approaches

doi: 10.7717/peerj.16166

Figure Lengend Snippet: (A) The expression level of CDT1 in pan-cancer (Blue: tumor tissue; Red: normal tissue). (B) The expression of CDT1 in LUAD and normal tissues (left: non-paired, n = 30; right: paired, n = 30) from the TCGA dataset (Blue: normal tissue; Red: tumor tissue). (C) The expression level of CDT1 in LUAD and normal tissues (paired and non-paired) from the GEO datasets (Blue: normal tissue; Red: tumor tissue. GSE118370 , n = 6; GSE140797 , n = 5; GSE31210 , normal sample, n = 20, tumor sample, n = 226). (D) Volcano plots of GEO datasets ( GSE118370 , GSE140797 and GSE31210 ) (Blue: down-regulated genes; Red: up-regulated genes) (* P < 0.05, *** P < 0.001). CDT1, chromatin licensing and DNA replication factor 1; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus; LUAD, lung adenocarcinoma.

Article Snippet: LUAD tissues and paired adjacent normal tissues were stained by immunohistochemistry with anti-human CDT1 (1:300; Rabbit; 14382-1-AP; Proteintech, Rosemont, IL, USA), followed by IVisionTM Poly-HRP goat anti-mouse/rabbit secondary antibody reagent (DD23-100; Xiamen Talent Biomedical Technology Co., Ltd., Xiamen, China) and DAB treatment.

Techniques: Expressing, Gene Expression

Journal: PeerJ

Article Title: Identification of CDT1 as a prognostic marker in human lung adenocarcinoma using bioinformatics approaches

doi: 10.7717/peerj.16166

Figure Lengend Snippet: (A) The Kaplan-Meier curve of OS in patients of different genders in TCGA cohort (Blue: male, n = 237; Red: female, n = 272). (B) The distribution of CDT1 expression in LUAD (Blue: CDT1 low, n = 92; Red: CDT1 high, n = 417). (C) Cumulative hazard for CDT1 high-expression and low-expression group (Blue: CDT1 low, n = 92; Red: CDT1 high, n = 417). (D) KM curves for overall survival (Blue: CDT1 low, n = 92; Red: CDT1 high, n = 417). (E) The distribution of CDT1 expression in GSE31210 (Blue: CDT1 low, n = 139; Red: CDT1 high, n = 86), and KM curves for overall survival (Blue: CDT1 low, n = 139; Red: CDT1 high, n = 86). (F) Nomogram based on CDT1 expression and clinicopathological factors (Including TCGA and GSE31210 , for TCGA, n = 509, for GSE31210 , n = 225). CDT1, chromatin licensing and DNA replication factor 1; OS, overall survival; TCGA, The Cancer Genome Atlas. For the survival: The shadows in Figure 2 represent the confidence intervals around the survival estimates. The shadows are typically shown as shaded areas around the plotted survival curves and provide information about the uncertainty associated with the survival estimates. “Number at risk” tables: The “Number at risk” tables accompanying the survival curves in Figure 2 represent the number of individuals or cases still under observation at each time point. For the Cumulative hazard: The cumulative hazard represents the accumulation of the hazard rates (instantaneous event rates) up to a certain point in time, the x-axis represents time, while the y-axis represents the cumulative hazard values. Each curve in the figure corresponds to a different subgroup or condition, allowing a visual comparison of the cumulative risk profiles. For nomogram: Nomogram is a graphical tool used for predicting a specific outcome or event based on multiple variables. The nomogram visually represents a mathematical model that combines the effects of different predictor variables to estimate a particular outcome. Each predictor variable is assigned a numerical value, and by drawing a vertical line from each variable’s value to the points axis, the corresponding points for each variable are determined.

Article Snippet: LUAD tissues and paired adjacent normal tissues were stained by immunohistochemistry with anti-human CDT1 (1:300; Rabbit; 14382-1-AP; Proteintech, Rosemont, IL, USA), followed by IVisionTM Poly-HRP goat anti-mouse/rabbit secondary antibody reagent (DD23-100; Xiamen Talent Biomedical Technology Co., Ltd., Xiamen, China) and DAB treatment.

Techniques: Expressing, Comparison

Journal: PeerJ

Article Title: Identification of CDT1 as a prognostic marker in human lung adenocarcinoma using bioinformatics approaches

doi: 10.7717/peerj.16166

Figure Lengend Snippet: (A) qPCR showed an increased level of CDT1 in LUAD and paired adjacent normal samples (Normal group: n = 16; Tumor group: n = 16). (B) H&E staining for LUAD and paired adjacent normal samples (Normal group: n = 16; Tumor group: n = 16). (C) Immunohistochemistry detecting the expression of CDT1 in LUAD and paired adjacent normal samples (Normal group: n = 16; Tumor group: n = 16) (** P < 0.01). CDT1, chromatin licensing and DNA replication factor 1; LUAD, lung adenocarcinoma; qPCR, quantitative real-time PCR; H&E, hematoxylin, and eosin.

Article Snippet: LUAD tissues and paired adjacent normal tissues were stained by immunohistochemistry with anti-human CDT1 (1:300; Rabbit; 14382-1-AP; Proteintech, Rosemont, IL, USA), followed by IVisionTM Poly-HRP goat anti-mouse/rabbit secondary antibody reagent (DD23-100; Xiamen Talent Biomedical Technology Co., Ltd., Xiamen, China) and DAB treatment.

Techniques: Staining, Immunohistochemistry, Expressing, Real-time Polymerase Chain Reaction

Journal: PeerJ

Article Title: Identification of CDT1 as a prognostic marker in human lung adenocarcinoma using bioinformatics approaches

doi: 10.7717/peerj.16166

Figure Lengend Snippet: (A) Volcano plot for DEGs between CDT1 high-expression and low-expression groups. (B) The GO and KEGG analysis for upregulated DEGs. (C) The heatmap for the top 50 genes that positively correlated with the expression of CDT1. (D) The GO and KEGG pathway analyses for CDT1 positive genes. CDT1, chromatin licensing and DNA replication factor 1; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, Differentially Expressed Genes.

Article Snippet: LUAD tissues and paired adjacent normal tissues were stained by immunohistochemistry with anti-human CDT1 (1:300; Rabbit; 14382-1-AP; Proteintech, Rosemont, IL, USA), followed by IVisionTM Poly-HRP goat anti-mouse/rabbit secondary antibody reagent (DD23-100; Xiamen Talent Biomedical Technology Co., Ltd., Xiamen, China) and DAB treatment.

Techniques: Expressing

Journal: PeerJ

Article Title: Identification of CDT1 as a prognostic marker in human lung adenocarcinoma using bioinformatics approaches

doi: 10.7717/peerj.16166

Figure Lengend Snippet: The correlation of CDT1 expression with the level of immune cell infiltration in LUAD. StromalScore: A computational algorithm that estimates the extent of stromal cell infiltration in tumor tissues. It quantifies the presence and abundance of stromal cells, which include fibroblasts, endothelial cells, and immune cells, within the tumor microenvironment. ImmuneScore: A computational metric that evaluates the level of immune cell infiltration in tumor tissues. It provides an estimation of the immune cell presence and activity within the tumor microenvironment. EstimateScore: A combined score that integrates both StromalScore and ImmuneScore. It represents the overall estimation of the stromal and immune components within the tumor, providing insights into the tumor microenvironment composition. Tumor purity: tumor purity refers to the proportion or percentage of cancer cells within the tumor sample. It represents the extent to which non-cancerous cells, such as stromal and immune cells, contribute to the overall tumor composition.

Article Snippet: LUAD tissues and paired adjacent normal tissues were stained by immunohistochemistry with anti-human CDT1 (1:300; Rabbit; 14382-1-AP; Proteintech, Rosemont, IL, USA), followed by IVisionTM Poly-HRP goat anti-mouse/rabbit secondary antibody reagent (DD23-100; Xiamen Talent Biomedical Technology Co., Ltd., Xiamen, China) and DAB treatment.

Techniques: Expressing, Activity Assay

Journal: PeerJ

Article Title: Identification of CDT1 as a prognostic marker in human lung adenocarcinoma using bioinformatics approaches

doi: 10.7717/peerj.16166

Figure Lengend Snippet: CDT1-related immune genes.

Article Snippet: LUAD tissues and paired adjacent normal tissues were stained by immunohistochemistry with anti-human CDT1 (1:300; Rabbit; 14382-1-AP; Proteintech, Rosemont, IL, USA), followed by IVisionTM Poly-HRP goat anti-mouse/rabbit secondary antibody reagent (DD23-100; Xiamen Talent Biomedical Technology Co., Ltd., Xiamen, China) and DAB treatment.

Techniques:

Journal: Cell Death Discovery

Article Title: Mitochondrial DNA leakage exacerbates odontoblast inflammation through gasdermin D-mediated pyroptosis

doi: 10.1038/s41420-021-00770-z

Figure Lengend Snippet: A Light microscopy examined the mDPC6T cells pyroptosis phenotype after transfected mtDNA (scale bar, 50μm). B LDH release assay was used to detect cell death after overexpression of mtDNA in mPDC6T cells (1 μg/well, 24 h). C The protein level of NLRP3, Caspase-1, GSDMD and IL-1β in mDPC6T cells was measured by western blotting after transfected mtDNA. D qPCR was performed to assess the mtDNA copy number in mDPC6T cells after EtBr treatment (1 μg/ml, 48 h). E The protein level of NLRP3, Caspase-1, GSDMD and IL-1β in mDPC6T cells after treatment with LPS and EtBr examined by immunoblotting. The data are the means ± standard deviations of 3 independent experiments ( p < 0.05 * , p < 0.01 ** , p < 0.001 *** ).

Article Snippet: The cells were treated with the indicated lipopolysaccharide (LPS, Sigma, USA) concentration at the indicated time, and the mitochondrial DNA replication inhibitor Ethidium Bromide (EtBr, Thermo Fisher Scientific, USA) was dissolved in DMEM to produce a final concentration of 1 μg/ml.

Techniques: Light Microscopy, Transfection, Lactate Dehydrogenase Assay, Over Expression, Western Blot

Journal: Cell Death Discovery

Article Title: Mitochondrial DNA leakage exacerbates odontoblast inflammation through gasdermin D-mediated pyroptosis

doi: 10.1038/s41420-021-00770-z

Figure Lengend Snippet: A The mRNA level of inflammatory cytokines, CXCL10 and IFN-β was detected by qPCR after overexpressing mtDNA (1 μg/well). B qPCR was performed to assess the CXCL10 and IFN-β mRNA level in mDPC6T cells after EtBr (1 μg/ml, 48 h) and LPS (20 μg/ml, 24 h) treatment. The data are the means ± standard deviations of three independent experiments ( p < 0.05 * , p < 0.01 ** , p < 0.001 *** ).

Article Snippet: The cells were treated with the indicated lipopolysaccharide (LPS, Sigma, USA) concentration at the indicated time, and the mitochondrial DNA replication inhibitor Ethidium Bromide (EtBr, Thermo Fisher Scientific, USA) was dissolved in DMEM to produce a final concentration of 1 μg/ml.

Techniques: