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The E2F8/NCAPG axis affects TAN phenotype and tumorigenesis in mice. KLN205 cells stably transfected with E2F8 KD and NCAPG OE were implanted into DBA/2 mice subcutaneously. (A) Growth rate of subcutaneous tumors in mice over 28 days. (B) Representative images and weight of in mice on day 28. (C-D) Populations of iNOS + Ly6G + (N1) and Arg1 + Ly6G + (N2) neutrophils in tumor tissues determined using double label immunofluorescence staining. (E) Concentrations of IL-8 and VEGFA in tumors determined using ELISA kits. (F) Levels of p-SMAD2 and p-SMAD3 in tumors determined using WB analysis. (G) Cell apoptosis in tumors determined using TUNEL assay. (H) Expression of E2F8 and NCAPG in tumor tissues analyzed using IHC. (I-K) Flow cytometry detection of the proportion of total CD3⁺CD45⁺CD8⁺ T cells (I), GZMB⁺ (J) or TNF-α⁺ CD8⁺ (K) T cells in tumor tissues. (L) KLN205 mouse lung cancer cells were subjected to E2F8 KD and NCAPG OE transfections and then implanted into DBA/2 mice via tail vein injection, followed by Kaplan-Meier analysis for 70-day survival rate of mice in each group. Data are expressed as the mean ± SD and shown as dot plots with whiskers representing SD. In vitro assays represent n = 6 independent biological replicates. For animal experiments, n = 6 biologically independent mice were analyzed per group, except n = 10 per group in panel L. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Journal: International Journal of Biological Sciences

Article Title: Targeting NCAPG with Arundanine Suppresses Neutrophil/NET-Mediated Immune Evasion and Enhances CD8 + T Cell Function in Lung Squamous Cell Carcinoma

doi: 10.7150/ijbs.134668

Figure Lengend Snippet: The E2F8/NCAPG axis affects TAN phenotype and tumorigenesis in mice. KLN205 cells stably transfected with E2F8 KD and NCAPG OE were implanted into DBA/2 mice subcutaneously. (A) Growth rate of subcutaneous tumors in mice over 28 days. (B) Representative images and weight of in mice on day 28. (C-D) Populations of iNOS + Ly6G + (N1) and Arg1 + Ly6G + (N2) neutrophils in tumor tissues determined using double label immunofluorescence staining. (E) Concentrations of IL-8 and VEGFA in tumors determined using ELISA kits. (F) Levels of p-SMAD2 and p-SMAD3 in tumors determined using WB analysis. (G) Cell apoptosis in tumors determined using TUNEL assay. (H) Expression of E2F8 and NCAPG in tumor tissues analyzed using IHC. (I-K) Flow cytometry detection of the proportion of total CD3⁺CD45⁺CD8⁺ T cells (I), GZMB⁺ (J) or TNF-α⁺ CD8⁺ (K) T cells in tumor tissues. (L) KLN205 mouse lung cancer cells were subjected to E2F8 KD and NCAPG OE transfections and then implanted into DBA/2 mice via tail vein injection, followed by Kaplan-Meier analysis for 70-day survival rate of mice in each group. Data are expressed as the mean ± SD and shown as dot plots with whiskers representing SD. In vitro assays represent n = 6 independent biological replicates. For animal experiments, n = 6 biologically independent mice were analyzed per group, except n = 10 per group in panel L. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The contents of tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), and vascular endothelial growth factor A (VEGFA) in TAN supernatant and tumor tissue homogenates were quantified using ELISA kits for Human IL-8 (E-EL-H6008, Elabscience Biotechnology Co., Ltd.), Human TNF-α (E-EL-H0109, Elabscience), Human VEGFA (E-EL-H0111, Elabscience), Mouse KC (IL-8, MBS8579382, Mybiosource, San Diego, CA, USA), and Mouse VEGFA (E-EL-M1292, Mybiosource).

Techniques: Stable Transfection, Transfection, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Analysis, TUNEL Assay, Expressing, Flow Cytometry, Injection, In Vitro