Journal: Genes & Diseases

Article Title: NKAPL suppresses NSCLC progression by enhancing the protein stability of TRIM21 and further inhibiting the NF-κB signaling pathway

doi: 10.1016/j.gendis.2025.101598

Figure Lengend Snippet: NKAPL interacts with the TRIM21 protein and increases its stability. (A) The Venn diagram illustrating the number of both specific and nonspecific NKAPL interactors. (B) KEGG pathway analysis of NKAPL-interacting proteins. (C) Correlation analysis of NKAPL and TRIM21 mRNA levels in NSCLC tissues. (D) The levels of TRIM21 were determined by western blotting analysis when NKAPL was overexpressed. (E) Co-immunoprecipitation-western blot analysis of A549 cells. The lysates were incubated with anti-GFP antibodies, and the fractionated immunoprecipitates were examined via western blotting with antibodies against TRIM21 and GFP. (F) A549 and H460 cells were transfected with a GFP-tagged NKAPL overexpression plasmid for 48 h. Immunofluorescence staining was performed using anti-TRIM21 antibodies, and the cell nuclei were stained with DAPI (blue). The subcellular localization of GFP (green) and TRIM21 (red) was observed via laser scanning confocal microscopy. (G) A549 cells were transfected with NKAPL-GFP for 48 h and then treated with the protein synthesis inhibitor cycloheximide (CHX; 100 μg/mL) for 0, 2, 4, 6, or 8 h. The expression levels of NKAPL and TRIM21 were measured via western blotting.

Article Snippet: The sections were incubated with primary antibodies against GFP (sc-9996; Santa Cruz) and Ki67 (sc-23900, 1:200 dilution).

Techniques: Western Blot, Immunoprecipitation, Incubation, Transfection, Over Expression, Plasmid Preparation, Immunofluorescence, Staining, Confocal Microscopy, Expressing

Journal: Genes & Diseases

Article Title: NKAPL suppresses NSCLC progression by enhancing the protein stability of TRIM21 and further inhibiting the NF-κB signaling pathway

doi: 10.1016/j.gendis.2025.101598

Figure Lengend Snippet: NKAPL inhibits the NF-κB signaling pathway through TRIM21. (A – C) Western blotting analysis of the expression of phosphorylated p65, p65, phosphorylated IKKα/β, phosphorylated IKBα, IKKα, IKKβ, and IKBα in (A) TRIM21 overexpression, (B) NKAPL-GFP overexpression, and (C) NKAPL-GFP-overexpression plus TRIM21 knockdown. (D) Immunofluorescence staining was performed with anti-p65 and anti-phosphorylated p65 antibodies, and the cell nuclei were stained with DAPI (blue). Subcellular localizations of GFP (green), p65 (red), and phosphorylated p65 (red) were observed via laser scanning confocal microscopy.

Article Snippet: The sections were incubated with primary antibodies against GFP (sc-9996; Santa Cruz) and Ki67 (sc-23900, 1:200 dilution).

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Immunofluorescence, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Selenoprotein P Deficiency Drives Hepatocellular Carcinoma Progression via Induction of Neutrophil Senescence and Immunosuppressive Microenvironment

doi: 10.1101/2025.06.24.661430

Figure Lengend Snippet: (a) Representative images of livers from S100a8-Cre and Lrp8 f/f ; S100a8-Cre ( Lrp8 ΔNeu ) mice at endpoint, demonstrating increased tumor burden in Lrp8 ΔNeu mice. (b, c) Quantification of liver-to-body weight ratio (b) and tumor counts (c) in Lrp8 ΔNeu versus control groups, showing a significantly higher tumor burden in the Lrp8 ΔNeu group (n = 9 per group). (d) Flow cytometry analysis showing an increased percentage of neutrophils in the tumor microenvironment of Lrp8 ΔNeu mice compared to controls (n = 6 per group). (e, f) Flow cytometric analysis of senescence markers in tumor-infiltrating neutrophils from represent mice. Senescence-associated β-galactosidase (SA-β-Gal) positive neutrophils (e) and p21 + neutrophils (f) were significantly increased in Lrp8 ΔNeu mice compared to control group (n = 5-6 per group). (g, h) Flow cytometric analysis of neutrophils isolated from Lrp8 ΔNeu and control tumors. Representative histogram showing NIR-H 2 SE fluorescence, a hydrogen selenide probe, in neutrophils. Quantification of the percentage of NIR-H 2 SE + neutrophils (g) and mean fluorescence intensity (MFI) of NIR-H 2 SE in neutrophils (h), showing significantly reduced hydrogen selenide levels in neutrophils after Lrp8 knockout (n = 5–6 per group). (i) Quantitative RT-PCR analysis of senescence-associated markers Cdkn1a in tumor-infiltrating neutrophils (n = 5–6 per group). (j, k) mRNA expression levels of S100a8 and S100a9 significantly elevated in tumor-infiltrating neutrophils from Lrp8 ΔNeu mice (n = 5-6 per group). (l) Schematic representation of hydrodynamic tail vein injection (HDTVi) constructs. Mice were injected with constructs encoding oncogenic NRas G12V and myr-AKT along with a plasmid that encodes a green fluorescent protein (GFP) -Sepp1 fusion protein for tracking Sepp1 localization. (m) Representative immunofluorescence images of liver tissues showing GFP (green) indicating Sepp1 localization, S100A9 (red) indicating neutrophils, lymphocyte antigen 6 family member G (Ly6G; white) as another neutrophil marker, and 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclei. Images demonstrate the colocalization of GFP with neutrophils, indicating that neutrophils uptake Sepp1. The lower row of images shows magnified views of the boxed regions, highlighting Sepp1 uptake by neutrophils. Data are presented as mean ± SEM; statistical significance was determined using two-tailed unpaired Student’s t-tests.

Article Snippet: Tumor sections were blocked with 1% bovine serum albumin (BSA) for 30 min at room temperature, after which sections were incubated (overnight at 4 °C) in PBS-1% BSA containing a primary antibody against green fluorescent protein (GFP; 1:500, Abmart, Cat. M20004), Ly6G (1:200, Servicebio, Cat. GB11229), or S100A9 (1:500, R&D Systems, Cat. AF2065).

Techniques: Control, Flow Cytometry, Isolation, Fluorescence, Knock-Out, Quantitative RT-PCR, Expressing, Injection, Construct, Plasmid Preparation, Immunofluorescence, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Kmt2c / d promotes gastric cancer initiation and confers vulnerability to mTORC1 inhibition and anti-PD1 immunotherapy

doi: 10.1101/2025.03.27.645747

Figure Lengend Snippet: A, Schematic of mouse models: two doses of tamoxifen (3 mg × 2) were injected intraperitoneally with a 48-hour interval. B, Representative immunofluorescence (IF) staining of EYFP in stomach tissues from Tmprss2-CreER T2 ;Rosa26-LSL-EYFP mice . Nuclei were counterstained with DAPI. Scale bar, 200 µm. C, Kaplan-Meier plots showing the survival of mice after gene knockout. D, Stomach weight of the indicated genetically engineered mouse model (GEMM) six weeks after tamoxifen injection. Data are presented as mean ± SD and analyzed with two-tailed t-test. E, Thickness in corpus mucosa measured by microscopy of hematoxylin and eosin (H&E) staining section. Each dot represents the averaged thickness of 5 random fields from one mouse. Data are presented as mean ± SD and analyzed with two-tailed t-test. F, Representative H&E staining of stomach tissues after tamoxifen administration. Scale bar, 200 µm. G, Pathological staging of stomach cancer progression based on infiltration of tumor cells in TPCD mice. H, Representative H&E staining of small and large intestines in TPCD mice. Scale bar, 1 mm.

Article Snippet: Immunofluorescent staining was performed using primary antibodies against EYFP (2956, Cell Signaling Technology, 1:100), H3K4me1 (5326, Cell Signaling Technology, 1:100), ATP4A (D031-3, MBL Life Science, 1:500), and Puromycin (MABE343, Millipore Sigma, 1:100).

Techniques: Injection, Immunofluorescence, Staining, Gene Knockout, Two Tailed Test, Microscopy

Journal: The EMBO Journal

Article Title: Slik sculpts the plasma membrane into cytonemes to control cell-cell communication

doi: 10.1038/s44318-025-00401-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Discs were fixed in PBS containing 0.2% Tween-20 (PBT) and 4% formaldehyde for 20 min. After several washes with PBT, samples were blocked in PBT with 0.1% BSA, followed by incubation overnight at 4 °C with primary antibodies against GFP (1:100, TP401; OriGene Technologies) (Fig. ).

Techniques: Stripping Membranes, Recombinant, Sequencing, Transfection, Luciferase, Software