Journal: Cell death discovery

Article Title: Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

doi: 10.1038/s41420-024-02019-x

Figure Lengend Snippet: Fig. 1 Nur77 inhibited ESCC cell proliferation in vitro and tumorigenesis in vivo. A, B qRT-PCR and western blot analysis of Nur77 expression in NEEC and ESCC cell lines (n = 3). C Overexpression of Nur77 in Kyse520 and TE-1 cell lines was analyzed by western blot. GAPDH was used as a loading control (n = 3). D The viability of ESCC cells overexpressing Nur77 was inhibited, as determined by the CCK-8 assay (n = 3). E The colony formation ability of ESCC cells overexpressing Nur77 was reduced (n = 3). F The percentage of Kyse520 and TE-1 cells that underwent Nur77 overexpression was increased (n = 3). G Western blotting was performed to investigate the expression levels of Bcl-2, Bax, cleaved caspase-3, and cleaved PARP in Kyse520 and TE-1 cells overexpressing Nur77. GAPDH was used as a loading control (n = 3). Representative tumor images (H), tumor volumes (I) and tumor weights (J) were collected from nude mice with tumor xenografts derived from TE-1 cells stably overexpressing Nur77 (n = 5). Nur77 protein expression in tissue samples from the xenografts was detected via immunohistochemistry (K) and western blotting (L) (n = 3). M Expression of cleaved caspase-3, Ki67, and PCNA in tumor xenografts was determined via immunohistochemistry (n = 3). Scale bars: 20 μm. All in vitro experiments were performed with three independent experiments. An unpaired Student’s t tests were used for statistical analysis, and the error bars indicate the means ± S.D. **P < 0.01 indicates a significant difference from the vector group.

Article Snippet: Using protein A/G magnetic beads (Cat# 88802, Thermo Fisher Scientific) and antibodies against Nur77 (1:200, Cat# NB10056745, Novus) or control rabbit IgG (1:100, Cat# 2729, Cell Signaling Technology), the nuclear lysate was incubated at 4 °C overnight on a rotator.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Control, CCK-8 Assay, Derivative Assay, Stable Transfection, Immunohistochemistry, Plasmid Preparation

Journal: Cell death discovery

Article Title: Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

doi: 10.1038/s41420-024-02019-x

Figure Lengend Snippet: Fig. 2 Silencing of Nur77 promoted tumor cell proliferation in vitro and in vivo. A Verification of Nur77 knockdown in Eca109 and Kyse510 cells by western blot analysis. GAPDH was used as a loading control (n = 3). Nur77 knockdown increased cell proliferation (B) and colony formation ability (C) (n = 3). D The percentage of Eca109 and Kyse510 cells undergoing apoptosis following Nur77 knockdown was decreased (n = 3). E Nur77 knockdown increased the expression of Bcl-2 and decreased the expression of Bax, cleaved caspase-3, and cleaved PARP. Cell lysates were assessed by western blotting (n = 3). F Images of tumor tissues collected from nude mice with stable Nur77 knockdown xenograft tumors derived from TE-1 cells (n = 5). Xenograft tumor growth was monitored (G) and weighed (H) (n = 5). Nur77 knockdown in tissue samples from the xenografts was evaluated via immunohistochemistry (I) and western blotting (J) (n = 3). K The expression of cleaved caspase-3, Ki67, and PCNA in tumor xenografts was determined by immunohistochemical analysis (n = 3). Scale bars: 20 μm. All in vitro experiments were performed with three independent experiments. An unpaired Student’s t-test was used for statistical analysis, and the error bars indicate the means ± S.D. **P < 0.01 indicates a significant difference from the shNC group.

Article Snippet: Using protein A/G magnetic beads (Cat# 88802, Thermo Fisher Scientific) and antibodies against Nur77 (1:200, Cat# NB10056745, Novus) or control rabbit IgG (1:100, Cat# 2729, Cell Signaling Technology), the nuclear lysate was incubated at 4 °C overnight on a rotator.

Techniques: In Vitro, In Vivo, Knockdown, Western Blot, Control, Expressing, Derivative Assay, Immunohistochemistry, Immunohistochemical staining

Journal: Cell death discovery

Article Title: Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

doi: 10.1038/s41420-024-02019-x

Figure Lengend Snippet: Fig. 4 IRF1 knockdown suppressed ESCC cell proliferation, and upregulating IRF1 in ESCC cells overexpressing Nur77 rescued the suppressive effects of Nur77 in vitro. Confirmation of IRF1 knockdown in Kyse520 and TE-1 cells by western blot (A, B) and qRT‒PCR (C). GAPDH was used as a loading control (n = 3). IRF1 knockdown decreased cell proliferation (D) and colony formation ability (E) (n = 3). F The percentage of Kyse520 and TE-1 cells undergoing apoptosis following IRF1 knockdown was increased (n = 3). G Western blot analysis showed that IRF1 knockdown decreased the expression of Bcl-2 and increased the expression of Bax, cleaved caspase-3, and cleaved PARP (n = 3). H Western blot of IRF1 protein expression in Nur77-overexpressing ESCC cells overexpressing IRF1. GAPDH was used as a loading control (n = 3). The influence of IRF1 overexpression on the inhibitory effects of Nur77 on cell growth (I) and colony formation (J, K) in vitro was detected by CCK-8 and colony formation assays (n = 3). The data are shown as the mean ± S.D. from experiments with three replicates. An unpaired Student’s t test was used for statistical analysis. **P < 0.01 indicates a significant difference compared with the vehicle group.

Article Snippet: Using protein A/G magnetic beads (Cat# 88802, Thermo Fisher Scientific) and antibodies against Nur77 (1:200, Cat# NB10056745, Novus) or control rabbit IgG (1:100, Cat# 2729, Cell Signaling Technology), the nuclear lysate was incubated at 4 °C overnight on a rotator.

Techniques: Knockdown, In Vitro, Western Blot, Control, Expressing, Over Expression, CCK-8 Assay

Journal: Cell death discovery

Article Title: Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

doi: 10.1038/s41420-024-02019-x

Figure Lengend Snippet: Fig. 7 Nur77 expression was negatively correlated with IRF1 expression in primary ESCC tissues. The mRNA expression of Nur77 (A) and IRF1 (B) in 72 paired ESCC and nontumor tissues was detected via qRT‒PCR. Nur77 (C) and IRF1 (D) expression levels in ESCC and nontumor tissues in the GSE38129, GSE45670, and GSE53625 datasets. E The correlation between Nur77 and IRF1 mRNA expression in 72 paired cancerous/noncancerous esophageal tissues from primary ESCC patients. The Pearson correlation coefficients (r) and p values are indicated. F The protein expression levels of Nur77, IRF1, and PD-L1 in eight paired ESCC and nontumor tissues were detected via western blotting (n = 3). G Representative images of IHC staining for the Nur77, IRF1, and PD-L1 proteins in cancerous and noncancerous esophageal tissues. Scale bars: 20 μm. H Kaplan‒Meier analysis of overall survival stratified by Nur77 and IRF1 expression in 72 ESCC patients. The log-rank test was used to determine statistical significance. The data are shown as the mean ± S.D. from experiments with three replicates. Paired Student’s t tests were used for statistical analysis. *P < 0.05, **P < 0.01 indicate significant differences from the normal group.

Article Snippet: Using protein A/G magnetic beads (Cat# 88802, Thermo Fisher Scientific) and antibodies against Nur77 (1:200, Cat# NB10056745, Novus) or control rabbit IgG (1:100, Cat# 2729, Cell Signaling Technology), the nuclear lysate was incubated at 4 °C overnight on a rotator.

Techniques: Expressing, Western Blot, Immunohistochemistry