cjun  (Cell Signaling Technology Inc)

Journal: Cell Death Discovery

Article Title: Atorvastatin improves spermatogenesis in murine and in vitro human chronic orchitis models through restoring blood-testis barriers

doi: 10.1038/s41420-025-02749-6

Figure Lengend Snippet: a Venn diagram of differential expressed proteins (DEPs) between groups. b Enriched GO terms of the co-downregulated proteins in Vehicle and T + AT groups compared with TNF-α group. c Heatmap showing the top 10 co-downregulated proteins according to the fold change between T + AT group and TNF-α group. d Western blot images of MMP3 and MMP9 in TM4 Sertoli cells from Vehicle, TNF-α and T + AT groups. e Grayscale value analysis of western blot for MMP3 and MMP9 in TM4 Sertoli cells from Vehicle, TNF-α and T + AT groups, n = 3. f , g Immunofluorescence of MMP3 and MMP9 (green) co-stained with SOX9 (red) in Vehicle, EAO-Sal and EAO-AT groups and quantitative analysis for immunofluorescence intensity of MMP3 and MMP9 in SOX9 + Sertoli cells from 20 randomly selected fields. SOX9 labeled Sertoli cells. Scale bar, 10 μm. n = 4 testis samples per group. h Western blot assay of Rac1, cJUN and Phospho-cJUN (p-cJUN) in TM4 Sertoli cells from Vehicle, TNF-α and T + AT groups. i Grayscale value analysis of western blot for Rac1 and p-cJUN in TM4 Sertoli cells from Vehicle, TNF-α and T + AT groups, n = 3. j Immunofluorescence of cJUN (green) co-stained with SOX9 (red) in Vehicle, EAO-Sal and EAO-AT groups and quantitative analysis for immunofluorescence intensity of cJUN in the nucleus of SOX9 + Sertoli cells from 20 randomly selected fields. SOX9 labeled Sertoli cells. Scale bar, 10 μm. k Western blot images of MMP3, MMP9, CX43 and ZO1 in TM4 Sertoli cells after T-5224 treatment. Data are presented as mean ± SEM, * P < 0.05, ** P < 0.01, **** P < 0.0001, ns indicates no statistical significance.

Article Snippet: The primary antibodies used in this study were listed: rabbit anti-HMGCR (A19063, ABclonal, 1:1000), rabbit anti-CX43 (26980-1-AP, Proteintech, 1:1000), rabbit anti-ZO1 (21773-1-AP, Proteintech, 1:1000), rabbit anti-CTNNB1 (ab32572, Abcam, 1:1000), rabbit anti-MMP3 (17873-1-AP, Proteintech, 1:1200), rabbit anti-MMP9 (10375-2-AP, Proteintech, 1:1200), rabbit anti-cJUN (ab32137, Abcam, 1:4000), rabbit anti-p cJUN (9261S, Cell Signaling Technology, 1:1000), rabbit anti-Rac1 (24072-1-AP, Proteintech, 1:1000).

Techniques: Western Blot, Immunofluorescence, Staining, Labeling

Journal: Cell reports

Article Title: Mutant RIT1 cooperates with YAP to drive an EMT-like lung cancer state

doi: 10.1016/j.celrep.2025.116185

Figure Lengend Snippet: (A) Growth in low attachment assay of isogenic SALE cells expressing RIT1 M90I and YAP1 8SA alone or in combination analyzed by CellTiterGlo. Data shown are the mean ± SD of 14–16 technical replicates. (B) Heatmap of gene expression data derived from bulk RNA-seq of isogenic SALE cells. Each row is a differentially expressed gene, and each column is a replicate of the cell line variant indicated. The top 100 up- and downregulated genes distinguishing combined RIT1 M90I /YAP1 8SA (RY) cells from YAP1 8SA cells were determined by marker selection based on the mean difference of RY replicates compared to YAP1 8SA replicates and then genes and samples were clustered using one minus Pearson correlation. (C) Volcano plot of differentially expressed genes from RNA-seq in SALE-RY cells versus parental SALE cells. p values shown were calculated by t tests of three biological replicates per cell line. Multiple hypothesis testing was performed using the false discovery rate (FDR) method. Red points: log 2 fold change (log 2 FC) > 1.5, false discovery rate (FDR) < 0.1; blue points: log 2 FC < −1.5, FDR < 0.1. (D) MSigDB overlap analysis of up- and downregulated genes from RNA-seq in RY cells compared to parental. The FDR is the false discovery rate analog of the hypergeometic p value after correction for multiple hypothesis testing. (E) Motif analysis of up- and downregulated genes from (C) using the MSigDB Transcription Factor Target (TFT) gene set. AP-1 and ZEB1 (AREB6) motifs are shown in bold. FDR was calculated as in (D). (F) Western blot of isogenic SALE cells using antibodies against RIT1 and AP-1 transcription factors cJUN and FOSL1/FRA1. Vinculin is used as a loading control. (G) Quantification of biological replicates ( n = 3) of cJUN abundance determined by western blot, normalized to loading control and parental cJUN abundance. Data shown are mean ± SEM. P, parental; R, RIT1 M90I ; Y, YAP1 8SA ; RY, RIT1 M90I/ YAP1 8SA . (H) Quantification of biological replicates ( n = 3) of FRA1 abundance determined by western blot, normalized to loading control and parental FRA1 abundance. Data shown are mean ± SEM. Labeling as in (G). See also and . ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by unpaired two-tailed t test.

Article Snippet: Primary antibodies were diluted in 50 μL 1% BSA PBST with 1:200 cJUN (60A8) (Cell Signaling Technology, 9165) and 1:50 YAP (Santa Cruz Biotechnology, sc-101199) and incubated at 4°C overnight.

Techniques: Expressing, Gene Expression, Derivative Assay, RNA Sequencing, Variant Assay, Marker, Selection, Western Blot, Control, Labeling, Two Tailed Test

Journal: Cell reports

Article Title: Mutant RIT1 cooperates with YAP to drive an EMT-like lung cancer state

doi: 10.1016/j.celrep.2025.116185

Figure Lengend Snippet: (A) Immunofluorescence and confocal images of SALE cells fixed and stained with anti-YAP and anti-cJUN antibodies and counterstained with DAPI. Scale bar, 100 μm. Inset, 50 μm. (B) Quantification of nuclear YAP fluorescence intensity determined by confocal microscopy. Data shown are mean ± SEM of four biological replicates. P, parental; R, RIT1 M90I ; Y, YAP1 8SA ; RY, RIT1 M90I /YAP1 8SA (C) Quantification of nuclear cJUN fluorescence intensity determined by confocal microscopy. Data shown are mean ± SEM of four biological replicates. Labeling as in (B). (D) Dual p-AP1 luciferase reporter assay in HEK293T cells transiently transfected with control vectors (V), RIT1 M90I (R), or YAP1 WT and YAP1 8SA in the presence or absence of RIT1 M90I . p-RL renilla luciferase was co-transfected and used for normalization. PMA was used as a positive control. Data shown are mean ± SEM of 3 technical replicates. Data are representative of at least three independent experiments. (E) Western blot of SALE-RY cells showing doxycycline-regulated induction of cJUN or a dominant-negative cJUN (TAM67). (F) In vivo xenograft tumor growth on doxycycline of the cells shown in (E). Data shown are mean ± SEM of 8–10 tumors per group. * P < 0.05 by one-way ANOVA. (G) Tumor weights of tumors shown in (F). Data shown are mean ± SEM. The cJUN wild-type group was not analyzed due to early euthanasia for tumor ulceration. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by unpaired two-tailed t test unless otherwise specified. See also .

Article Snippet: Primary antibodies were diluted in 50 μL 1% BSA PBST with 1:200 cJUN (60A8) (Cell Signaling Technology, 9165) and 1:50 YAP (Santa Cruz Biotechnology, sc-101199) and incubated at 4°C overnight.

Techniques: Immunofluorescence, Staining, Fluorescence, Confocal Microscopy, Labeling, Luciferase, Reporter Assay, Transfection, Control, Positive Control, Western Blot, Dominant Negative Mutation, In Vivo, Two Tailed Test

Journal: Cell reports

Article Title: Mutant RIT1 cooperates with YAP to drive an EMT-like lung cancer state

doi: 10.1016/j.celrep.2025.116185

Figure Lengend Snippet: (A) Western blot confirmation of short hairpin RNA (shRNA) knockdown of RIT1, Yap, and cJun. β-actin was used as a loading control. (B) Cell proliferation analysis using Incucyte imaging of S-RPN-1 cells with stable knockdown of each gene using the shRNAs indicated. Data shown are the mean ± SEM of three independent transductions. (C) Western blot of Zeb1 expression in S-RPN-1 cells with or without cJun knockdown. Actin was used as a loading control. (D) Western blot quantification of Zeb1 expression in cells after cJun knockdown. Data shown are mean ± SEM of three biological replicates. *, p < 0.05 by unpaired two-tailed t test. (E) Growth-in-low-attachment assay of SALE-RY cells in the presence and absence of 0.1 μM trametinib (MEKi) or increasing dosages of the pan-TEAD autopalmitoylation inhibitor VT104 (0.1, 0.25, 0.6, 1.6, 4, and 10 μM). Cells were plated in low attachment plates and incubated with each drug for 7 days before cell growth was determined by CellTiterGlo. The data shown are the mean and SEM of three technical replicates. **, P < 0.01; ****, P < 0.0001 by unpaired two-tailed t test. (F) In vitro dose response assay of S-RPN-1 tumor cells with trametinib (MEKi) or the TEAD1-selective inhibitor VT103, pan-TEAD inhibitors VT104 and VT107, and the less-active pan-TEAD inhibitor enantiomer VT106. Cells were plated in 96-well plates and analyzed by CellTiterGlo after 7 days of drug treatment. The data shown are the mean and SEM of three independent biological replicates. (G) IC50 of compounds tested in (B). The data shown are the mean and SEM of the IC50 determined from three independent experiments. ns, not significant. **, P < 0.01 by unpaired two-tailed t test. (H) Allograft tumor growth of S-RPN-1 cells in the flanks of immunocompromised mice ( n = 5 per group). Mice were enrolled in the study when tumor reached >70 mm 3 , after which animals were randomized to the treatment groups indicated. See for dosing details. Data shown are mean ± SEM. ns, not significant. ****, P < 0.0001 by one-way ANOVA with multiple testing correction. (I) Kaplan-Meier survival analysis of progress-free survival (PFS) of the mice shown in (D). PFS was defined as a 300% tumor volume increase. **, P < 0.01 by log-rank test. (J) MicroCT imaging of an S-RPN mouse at baseline (left), tumor initiation (middle), and after treatment (right) with VT104/MEKi combination therapy (20 mg/kg VT104 and 0.5 mg/kg trametinib). H, heart. (K) Tumor nodule volume comparison of untreated mice (gray) and two nodules from the mouse shown in (J) (red) over a 2–4 week time period. See also .

Article Snippet: Primary antibodies were diluted in 50 μL 1% BSA PBST with 1:200 cJUN (60A8) (Cell Signaling Technology, 9165) and 1:50 YAP (Santa Cruz Biotechnology, sc-101199) and incubated at 4°C overnight.

Techniques: Western Blot, shRNA, Knockdown, Control, Imaging, Expressing, Two Tailed Test, Incubation, In Vitro, Comparison