Journal: Biochemistry and Biophysics Reports

Article Title: Bifendate inhibits cell PARthanatos by activating the MEK/ERK pathway

doi: 10.1016/j.bbrep.2026.102454

Figure Lengend Snippet: DDB sustains cellular phospho-ERK levels. (A–B) HeLa cells were pre-treated with DDB for 24 h, exposed to MNNG (60 μM, 15 min) and cultured for a further 8 h. ERK1 and phospho-ERK1/2 expression were analysed by immunoblot (A) and densitometry (B). (C) Cells pre-treated with DDB for 24 h were challenged with MNNG (60 μM, 15 min) and harvested 4 h later; MEK1/2 and phospho-MEK1 levels were analysed by immunoblot. (D) The thermal stability of MEK and ERK after 24 h pretreatment with DDB (25 μM) or DMSO was detected by Western blot. (E) The grey values of each protein band were analysed quantitatively. (n = 3). The values are expressed as the means ± SDs. Statistical analysis was performed using T-test. *p < 0.05. (F) The thermal stability of DUSP1 after 24 h pretreatment with DDB (25 μM) or DMSO was detected by Western blot. (G) The grey values of protein band were analysed quantitatively. (n = 3). The values are expressed as the means ± SDs. Statistical analysis was performed using T-test. (H) Cells were pre-treated or not with DDB for 24 h, exposed to MNNG (60 μM, 15 min) and cultured for 4 h then phospho-Bad expression was analysed. (I) Mitochondrial membrane potential was assessed by JC-1 staining after the indicated treatments. Scale bar: 50 μm. The values are expressed as the means ± SDs (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey's test. **p < 0.01.

Article Snippet: After sealing with 5 % skimmed milk, the membrane was incubated successively with the primary antibody (PAR/pADPr, R&D, #4335-MC-100; PARP1, ABclonal, #A0010; AIF, Santa Cruz Biotechnology, #sc-13116; TOMM20, Beyotime, #AF1717; MIF, Beyotime, #AF7467; β-Tubulin, Beyotime, #AF2839; ERK1, Beyotime, #AF1315; Phospho-Erk1 (Thr202/Tyr204)/Erk2 (Thr185/Tyr187), Beyotime, #AF1891); MEK1/2, Beyotime, #AF1057; Phospho-MEK1 (Ser218/222), Beyotime, #AF1786; DUSP1, MCE, #HY- P80645 ; GAPDH, ABclonal, # AC002) and the secondary antibody (ABclonal, #AS014; ABclonal, #AS003).

Techniques: Cell Culture, Expressing, Western Blot, Membrane, Staining

Journal: The FASEB Journal

Article Title: DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis

doi: 10.1096/fj.201802558rr

Figure Lengend Snippet: Figure 1. DUSP22 expression levels are significantly reduced in human and mouse PCa tissues. A, B) Analysis of DUSP22 expression was performed using microarray data obtained from GEO databases GSE6919 (A) and GSE21034 (B). DUSP22 expression is significantly lower in PCa tissues compared with normal tissues. C) Prostate tissues of 30-wk-old TRAMP mice and WT littermates were harvested for histology processing and detected with an anti-DUSP22 antibody. In WT (left panel) and TRAMP (middle and right panels) prostate glands, red arrows indicated regions of strong DUSP22-stained epithelial cells (brownish signals). The black arrow showed that fewer epithelial cells had DUSP22 expression within low-grade prostate intraepithelial neoplasia (PIN). DUSP22 expression was barely detectable in poorly differentiated adenocarcinoma (right panel) obtained from 33-wk-old TRAMP mice. Immunohistochemical staining images of DUSP22 shown here are representative micrographs (n = 3 for WT and TRAMP), magnified 310 and 40 (red box areas as indicated). Scale bar, 200 mm.

Article Snippet: DUSP22 antibody was from Proteintech (1:200; Rosemont, IL, USA).

Techniques: Expressing, Microarray, Staining, Immunohistochemical staining

Journal: The FASEB Journal

Article Title: DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis

doi: 10.1096/fj.201802558rr

Figure Lengend Snippet: Figure 2. DUSP22 expression in PCa cells inhibits cell growth and colony formation. A) LNCaP and C4-2B cells (5 3 103/well) stably transfected with parental vectors or DUSP22 were seeded in 96-well plates with regular medium for cell growth assay, as indicated. B) The colony formation assay was performed on LNCaP (left) and C4-2B (right) cells. The data shown (upper) are representative of 3 independent experiments in triplicates. Images (middle) of cells transfected with parental vectors or DUSP22 are indicated. Results are expressed as means 6 SD (n = 3). Student’s t tests were used for statistical analysis. C) C4-2B cells (1 3 106) were subcutaneously injected into athymic mice. The growth of C4-2B tumors was monitored weekly by manual caliper measurements. Each symbol represents a group mean of mice (n = 4–5), and error bars indicate SD. Cell and tumor growth curves were analyzed by ANOVA. P , 0.05 by Student’s t test or ANOVA indicates a statistically significant difference. **P , 0.01, ***P , 0.001.

Article Snippet: DUSP22 antibody was from Proteintech (1:200; Rosemont, IL, USA).

Techniques: Expressing, Stable Transfection, Transfection, Growth Assay, Colony Assay, Injection

Journal: The FASEB Journal

Article Title: DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis

doi: 10.1096/fj.201802558rr

Figure Lengend Snippet: Figure 3. In vitro screening of DUSP22 downstream targets using antibody array analysis. A) Representative blots of human oncology antibody array probed with protein lysates isolated from LNCaP cells carrying parental vectors (upper) or DUSP22 (lower). Red squares indicate a noticeable decrease in the level of array proteins when DUSP22 is expressed. B) PSA protein levels were reduced when DUSP22 was expressed in LNCaP cells, and this down-regulation occurred in a DUSP22 protein phosphatase-dependent manner. Actin was used as a loading control. Representative blots from duplicate experiments are shown. n.s, nonspecific binding. C) LNCaP cells (left panel) and C4-2B (right panel) were stably transfected with parental vectors, DUSP22, or DUSP22-Myc. PSA mRNA levels were determined by quantitative PCR using PSA-specific primers. Expression levels were expressed as fold-decrease relative to vector controls. D) Extracellular PSA levels in the conditioned medium of LNCaP cells with parental vectors or DUSP22 (61 nM of DHT stimulation) were measured using a chemiluminescent microparticle immunoassay. Data were obtained from 2 independent ELISA experiments run in triplicate. **P , 0.01, ***P , 0.001.

Article Snippet: DUSP22 antibody was from Proteintech (1:200; Rosemont, IL, USA).

Techniques: In Vitro, Ab Array, Isolation, Control, Binding Assay, Stable Transfection, Transfection, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis

doi: 10.1096/fj.201802558rr

Figure Lengend Snippet: Figure 4. Contrasting effects of DUSP22 expression on cell growth in different PCa cells. PC3 (A), DU145 (B), and CWR22Rv1 (C) cells (5 3 103/well) stably transfected with parental vectors, DUSP22, or DUSP22-Myc were seeded in 96-well plates with regular medium for cell growth assay, as indicated in Fig. 2A, B. Growth of PCa cells (vector vs. DUSP22) was examined using a Cell Counting Kit-8 (left panels). The colony formation assay was performed on PC3, DU145, and CWR22Rv1 cells (right panels). The data shown are representative of 2 independent experiments. Results are expressed as means 6 SD (n = 3). Ns, not significant. P , 0.05 by ANOVA indicates a statistically significant difference. **P , 0.01, ***P , 0.001.

Article Snippet: DUSP22 antibody was from Proteintech (1:200; Rosemont, IL, USA).

Techniques: Expressing, Stable Transfection, Transfection, Growth Assay, Plasmid Preparation, Cell Counting, Colony Assay

Journal: The FASEB Journal

Article Title: DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis

doi: 10.1096/fj.201802558rr

Figure Lengend Snippet: Figure 5. DUSP22 decreases EGFR and ERK1/2 phosphorylation. A) Representative blots of human p-RTK antibody array probed with protein lysates isolated from LNCaP cells carrying parental vectors (upper) or DUSP22 (lower). Red squares indicate decreased levels of array proteins when DUSP22 is expressed. B, C) Impact of DUSP22 expression on p-EGFR (Tyr1068) and EGFR levels in LNCaP (B) and C4-2B (C) cells, with or without EGF stimulation. D) Lysates from LNCaP cells (6DUSP22), with or without EGF stimulation, were immunoblotted with antibodies for total or phosphorylated forms of ERK1/2. Representative blots from duplicate experiments are shown.

Article Snippet: DUSP22 antibody was from Proteintech (1:200; Rosemont, IL, USA).

Techniques: Phospho-proteomics, Ab Array, Isolation, Expressing

Journal: The FASEB Journal

Article Title: DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis

doi: 10.1096/fj.201802558rr

Figure Lengend Snippet: Figure 7. The impact of DUSP22 expression on EGF-induced AR phosphorylation. A) DUSP22 expression decreases AR tyrosine phosphorylation (Tyr534) induced by EGF in LNCaP (upper panel) and C4-2B cells (lower panel). B) Total cell lysates from LNCaP cells containing c-Myc–tagged DUSP22 proteins were coimmunoprecipitated with an anti–c-Myc or anti-IgG control antibody, followed by immunoblotting with an anti-AR antibody, or with an anti–c-Myc antibody as a DUSP22 control. Total cell lysates (TCL) were also immunoblotted with the antibodies listed above. IB, immunoblot; IP, immunoprecipitation. C) Model for DUSP22-mediated suppression on EGFR and AR signaling in PCa cells through dephosphorylation.

Article Snippet: DUSP22 antibody was from Proteintech (1:200; Rosemont, IL, USA).

Techniques: Expressing, Phospho-proteomics, Control, Western Blot, Immunoprecipitation, De-Phosphorylation Assay