Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: ATF3 is predicted as a favorable prognostic factor and is positively associated with GDF15 in bladder cancer. (A) Data of TCGA gained from the GEPIA database showed ATF3 expression levels across 31 kinds of tumor samples and paired normal tissues. The bar height represents the median expression of ATF3 in certain tumor types or normal tissues, as indicated. (B) Expression levels of ATF3 in the TCGA database from 28 normal and 404 tumor groups, respectively. (C) Correlation between ATF3 expression levels and progression-free survival (P.F.S.) of bladder urothelial carcinoma patients generated from TCGA-BLCA database. (D) The co-expression of ATF3 with GDF15 in TCGA-BLCA database. (E) Kaplan-Meier analysis of progression-free survival of TCGA-BLCA database, comparing subsets with double high expressions of ATF3 and GDF15 to those with double low expressions. Abbreviation: ACC: adrenocortical carcinoma; BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: esophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LAML: acute myeloid leukemia; BLGG: brain lower grade glioma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: testicular germ cell tumors; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; and UCS: uterine carcinosarcoma. ∗, p < 0.05.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Generated

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of prognostic factors and ATF3 RNA expression in bladder cancer (N = 169).

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: RNA Expression, Biomarker Discovery, Expressing

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of prognostic factors and GDF15/ATF3 RNA expression in bladder cancer (N = 89).

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: RNA Expression, Biomarker Discovery, Expressing

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Modulation of ATF3 on cell growth and gene expression in bladder cancer cells. (A, left) The expressions of ATF3, GDF15, NDRG1, KAI-1, and β-actin of ectopic ATF3-overexpressed T24 (T24-ATF3) and mock-overexpressed T24 (T24-DNA) cells were determined by immunoblot assays. (A, right) Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin relative to the mock-control group (±SE; n = 3). Cell growth rate of T24-DNA and T24-ATF3 cells was determined by (B) Ki67 or (C) colony formation assays ( n = 3). (D, top) The expressions of ATF3, GDF15, NDRG1, KAI-1, and β-actin of ATF3-knockdown (HT_shATF3) or mock-knockdown (HT_shCOL) HT1376 cells were determined by immunoblot assays. (D, bottom) Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin relative to the mock-control group (±SE; n = 3). (E) The mRNA ratio of ATF3, GDF15, NDRG1, and KAI-1 between HT_shATF3 and HT_shCOL cells was determined by RT-qPCR. (F) The reporter activities of GDF15, NDRG1, and KAI-1 reporter vectors after transient overexpression of various dosages of the ATF3 expression vector, as indicated, were determined by reporter assays ( n = 6). (G) Cell growth rate following ATF3 knockdown was determined by Ki67 proliferation assays ( n = 3). ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Gene Expression, Western Blot, Produced, Control, Knockdown, Quantitative RT-PCR, Over Expression, Expressing, Plasmid Preparation

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Modulation of ATF3 on cell invasion and epithelial-to-mesenchymal transition. The invasion ability of (A) T24-DNA, T24-ATF3, (B) HT_shCOL, and HT_shATF3 cells was determined by Matrigel invasion assays. The quantitative analysis data were expressed as average cell counts/9 fields ± SE. (C) The expressions of N-cadherin, E-cadherin, Snail, Slug, and β-actin in HT_shCOL and HT_shATF3 cells were determined by immunoblot assays. (D) Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin relative to the mock-control (HT_shCOL) group (±SE; n = 3). (E) The F-actin staining with Texas Red X-Phalloidin and the fluorescence were recorded using a confocal microscope. (F) The intensities were measured along the line from the peripheral to the central area of the cells, and the quantitative analysis (G) of the F-actin fluorescence intensity of HT_shCOL and HT_shATF3 cells (±SE, n = 4). ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Western Blot, Produced, Control, Staining, Fluorescence, Microscopy

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Modulation of ATF3 on tumor growth of bladder cancer cells in xenograft model. (A) HT_shCOL and HT_shATF3 cells were injected subcutaneously in the dorsal area of the four-week-old male athymic nude mice (n = 8). Tumors derived from both cells were recorded after the mice were sacrificed. The tumor growth rates (B) and animal body weights (C) were measured within 24 days. (D) The tumor weights were recorded immediately after the sacrifice (±SE; n = 8). (E) The protein levels of ATF3, GDF15, NDRG1, KAI-1, and β-actin of the tumors derived from the HT_shCOL and HT_shATF3 cells were determined by immunoblot assays. (F) The quantitative analysis was presented as the relative density of the target proteins/β-actin (±SE; n = 5). ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Injection, Derivative Assay, Western Blot

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Modulation of metformin on the expressions of ATF3 and GDF15 in the bladder cancer cells. The expressions of ATF3, GDF15, NDRG1, and β-actin in the T24 cells after being treated with or without 4 mM of metformin at normal glucose conditions (5 mM) were determined by immunoblot assays (A) and quantitative analysis (B). The expressions of ATF3 and GDF15 in the HT1376 cells after being treated with 5 mM or 30 mM glucose and with/without 4 mM of metformin, as indicated. were determined by immunoblot assays (C) and quantitative analysis (D). (E) Gene expressions of ATF3 and GDF15 in HT1376 cells after being treated with/without various dosages of metformin, as indicated, were determined by RT-qPCR. Protein expressions of ATF3 and GDF15 in HT1376 cells after being treated with/without metformin or SB431542, as indicated, were determined by immunoblot assays (F) and quantitative analysis (G). (H) Protein expressions of ATF3 and GDF15 in HT_shCOL and HT_shATF3 cells after being treated with/without metformin were determined by immunoblot assays (left) and quantitative analysis (right). Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin (±SE; n = 3) relative to the vehicle-treated group. ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Western Blot, Quantitative RT-PCR, Produced

Journal: Biomedical Journal

Article Title: Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer

doi: 10.1016/j.bj.2024.100756

Figure Lengend Snippet: Co-modulation between ATF3 and GDF15 in the bladder cancer cells. The expressions of ATF3, GDF15, and β-actin in (A) T24-DNA, T24-GDF15, (B) HT_shCOL, and HT_shGDF15 cells were determined by immunoblot assays. Quantitative analysis data were expressed as the intensity of protein bands produced from the expressions of the target proteins/β-actin (±SE; n = 3) relative to the vehicle-treated group. The ratio of gene expressions of ATF3 and GDF15 in (C) T24-DNA, T24-GDF15, (D) HT_shCOL, HT_shGDF15, (E) T24-DNA, T24-ATF3, (F) HT_shCOL, and HT_shATF3 cells were determined by RT-qPCR. Data from quantitative analysis were expressed as the expressions of the target genes/β-actin relative to the mock-control group (±SE; n = 3). ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: GDF15 and ATF3 shRNA lentiviral transduction particles (sc-39335-V and sc-29757-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Western Blot, Produced, Quantitative RT-PCR, Control

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein induced type I collagen processing in conditioned media of human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 24 h, and conditioned media were collected and concentrated for Western blotting. Stain-free gel bands from corresponding cell lysate samples were used as the loading control. Representative Western blots of ( A ) type I procollagen and pC-collagen (tropocollagen with PICP), detected using anti-C-telo antibody (n = 16), ( B ) type I procollagen, pC-collagen (tropocollagen with PICP), and PICP, detected using anti-PICP antibody (n = 8), and ( C ) type I procollagen, detected using anti-PINP antibody (n = 8). Schematic molecular structures and approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Cell Culture, Recombinant, Western Blot, Staining

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: Silencing ADAMTS-2 inhibited WISP-1 protein-induced type I collagen processing in conditioned media of human cardiac fibroblasts (HCFs). HCFs were either transfected with control SiRNA (1.228 μM), ADAMTS SiRNAs (614 nM/target gene), or left untransfected prior to seeding on a 12-well plate. After culture in supplemented fibroblast growth medium for 24 h, HCFs were starved in serum-free medium (SFM) for 48 h. The medium was then replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) and HCFs cultured for 15 h for qPCR analysis, and 24 h or 96 h for Western blotting analysis. ( A ) Quantification of ADAMTS-2 mRNA expression using qPCR analysis. Data were normalised to 36B4 housekeeping gene and expressed as the relative fold change to the untransfected HCFs (Control). ( B ) Quantification of ADAMTS-2 protein expression (168 h post-transfection) using Western blotting analysis. Data were normalised to stain-free gel bands and expressed as the relative fold change to the untransfected HCFs (Control). ( C ) Representative Western blots of type I procollagen and pC-collagen (tropocollagen with PICP) detected using anti-C-telo antibody. Stain-free gel bands from corresponding cell lysate samples were used as loading control. Quantification of pC-collagen I protein expression (96 h post-transfection) was expressed as the relative fold change to the WISP-1 protein treatment group. Data shown as mean ± SEM (n = 4–6). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Transfection, Recombinant, Cell Culture, Western Blot, Expressing, Staining

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Cell Culture, Recombinant, Lysis, Western Blot, Expressing, MANN-WHITNEY, Incubation, Blocking Assay

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein promoted human cardiac fibroblasts (HCFs) activation. HCFs were cultured on soft substrate plates (8 kPa) in supplemented fibroblast growth medium for 24 h. HCFs were starved in serum-free medium (SFM) for 48 h, then the medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) and cultured for 24 h. ( A ) HCFs were fixed for immunocytochemical staining with anti-α-SMA antibody. α-SMA positive cells are stained green, and nuclei are stained blue with DAPI (4′,6-diamidino-2-phenylindole). Some positive cells are indicated by white arrows. Scale bar represents 50 μm. Quantification of positive α-SMA staining was expressed as the relative fold change to the control of the percentage of positive α-SMA staining cells to total cells on soft substrate. Data shown as mean ± SEM (n = 8). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Quantification of α-SMA protein expression and ( C ) quantification of PCNA protein expression using Western blotting analysis. Data were normalised to stain-free gel bands and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( D ) Quantification of accumulated migration distance per cell over the duration of consecutive images (21 h 30 min). Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Student’s t test. * indicates p < 0.05.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Activation Assay, Cell Culture, Recombinant, Staining, MANN-WHITNEY, Expressing, Western Blot, Migration

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 deficiency attenuated angiotensin II (AngII)-induced coronary artery perivascular fibrosis. Cardiac fibrosis was induced by subcutaneous AngII infusion (1000 ng/kg/min) for 28 days via osmotic pumps in WISP-1 +/+ and WISP-1 −/− mice. Representative images showing type I collagen (dark brown) staining using anti-C-telo antibody in left ventricular tissues with and without AngII infusion. Nuclei are stained blue with haematoxylin. Non-immune IgG was used as the negative control. Quantification of positive type I collagen staining was expressed as the percentage of positive collagen I staining area to total tissue area. Data shown as mean ± SEM (n = 5–8). Red arrows indicate some positive staining (dark brown). Scale bar represents 100 μm. Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Staining, Negative Control

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: A schematic summary of the findings of this study. WISP-1 promotes cardiac fibroblasts’ phenotypic switch from quiescent fibroblasts to myofibroblasts (activated fibroblasts), promoting collagen processing and accumulation. WISP-1 activates Akt signalling via integrin β1/FAK/ILK in cardiac fibroblasts. Deletion of WISP-1 attenuates angiotensin II (AngII)-induced cardiac fibrotic remodelling in vivo. Figure key is illustrated on the top left-hand side of the figure. Purple ↓ denotes promotion; black ↑ denotes increase; ┤ denotes inhibition.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: In Vivo, Inhibition