Journal: Scientific reports

Article Title: DCDC2 inhibits hepatic stellate cell activation and ameliorates CCl 4 -induced liver fibrosis by suppressing Wnt/β-catenin signaling.

doi: 10.1038/s41598-024-59698-w

Figure Lengend Snippet: Figure 1. Low expression of DCDC2 in human and mouse liver fibrotic tissues. (A) Hematoxylin and eosin (H&E), Sirius red staining and immunohistochemistry of DCDC2 were performed in tissues from human healthy controls and liver fibrosis samples. (B) The mRNA expression level of DCDC2 was measured using qPCR in human healthy control and liver fibrosis samples. (C) The protein expression levels of DCDC2, α-smooth muscle actin (α-SMA), and type I collagen alpha 1 (Col1α1) were assessed via western blot analysis in human healthy control and liver fibrosis samples. (D) H&E, Sirius red staining, Masson trichrome staining and immunohistochemistry analyses were conducted on liver tissues obtained from mice. (E) The protein expression levels of DCDC2, α-SMA, and Col1α1 were assessed via western blot analysis in liver tissues obtained from mice. (F) Immunofluorescence staining of DCDC2 (green) and α-SMA (red) in liver tissues from mice. Scale bar, 50 µM. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with Control, * P < 0.05.

Article Snippet: After blocking by 5% nonfat milk, the membranes were incubated with the primary antibody against the following targets: DCDC2 (26,978–1-AP, Proteintech, Wuhan, China), α-SMA (ab124964, Abcam, England), Col1α1 (AF6524, Beyotime Biotech, Shanghai, China), β-catenin (GB12015, Servicebio, Wuhan, China), CylcinD1 (26,939–1-AP, Proteintech, Wuhan, China), c-myc (10,828–1-AP, Proteintech, Wuhan, China), Lamin A/C (10,298–1-AP, Proteintech, Wuhan, China), E-cadherin (20,874–1-AP, Proteintech, Wuhan, China), N-cadherin (22,018–1-AP, Proteintech, Wuhan, China), Vimentin (10,366–1-AP, Proteintech, Wuhan, China), Snail1 (13,099–1-AP, Proteintech, Wuhan, China) and GAPDH (60,004-1-Ig, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Expressing, Staining, Immunohistochemistry, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: DCDC2 inhibits hepatic stellate cell activation and ameliorates CCl 4 -induced liver fibrosis by suppressing Wnt/β-catenin signaling.

doi: 10.1038/s41598-024-59698-w

Figure Lengend Snippet: Figure 2. Downregulation of DCDC2 in HSC activated by TGF-β1. (A) LX-2 cells were treated with different concentrations of TGF-β1 for 24 h. The protein expression levels of α-SMA and Col1α1 were detected using western blot. (B) LX-2 cells were treated with 10 ng/mL TGF-β1 at different time points. The protein expression levels of DCDC2, α-SMA and Col1α1 were assessed using western blot analysis. (C) The mRNA levels of DCDC2, α-SMA and Col1α1 were measured using qPCR. (D) Immunofluorescence staining of DCDC2 (green) and α-SMA (red) in LX-2 cells at 0 and 24 h following treatment with 10 ng/mL TGF-β1. (E) The efficiency of DCDC2 overexpression or silencing was measured by western blot. Scale bar, 50 µM. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with TGF-β1 0 ng/mL group (A) or TGF-β1 10 ng/mL 0 h group (D), * P < 0.05.

Article Snippet: After blocking by 5% nonfat milk, the membranes were incubated with the primary antibody against the following targets: DCDC2 (26,978–1-AP, Proteintech, Wuhan, China), α-SMA (ab124964, Abcam, England), Col1α1 (AF6524, Beyotime Biotech, Shanghai, China), β-catenin (GB12015, Servicebio, Wuhan, China), CylcinD1 (26,939–1-AP, Proteintech, Wuhan, China), c-myc (10,828–1-AP, Proteintech, Wuhan, China), Lamin A/C (10,298–1-AP, Proteintech, Wuhan, China), E-cadherin (20,874–1-AP, Proteintech, Wuhan, China), N-cadherin (22,018–1-AP, Proteintech, Wuhan, China), Vimentin (10,366–1-AP, Proteintech, Wuhan, China), Snail1 (13,099–1-AP, Proteintech, Wuhan, China) and GAPDH (60,004-1-Ig, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Over Expression

Journal: Scientific reports

Article Title: DCDC2 inhibits hepatic stellate cell activation and ameliorates CCl 4 -induced liver fibrosis by suppressing Wnt/β-catenin signaling.

doi: 10.1038/s41598-024-59698-w

Figure Lengend Snippet: Figure 3. DCDC2 inhibits the activation of HSC induced by TGF-β1 by targeting the Wnt/β-catenin pathway. (A) The protein expression levels of β-catenin, α-SMA, and Col1α1 were assessed via western blot analysis subsequently to transfecting LX-2 cells with DCDC2 overexpressing lentiviral vector and negative control vector, followed by stimulation with TGF-β1. (B) The protein expression levels of β-catenin, α-SMA, and Col1α1 were evaluated through western blot analysis after transfecting LX-2 cells with DCDC2-sgRNA and negative control sgRNA, followed by stimulation by TGF-β1. (C) The protein expression levels of β-catenin, cyclinD1, and c-myc were detected by western blot after LX-2 cells were transfected with DCDC2 overexpressing lentiviral vector and/or then stimulated by SKL2001. (D) The protein expression levels of β-catenin, cyclinD1, and c-myc were assessed via western blot analysis after transfecting LX-2 cells with DCDC2-sgRNA and/or then stimulated by ICG001. (E) The protein expression levels of β-catenin in the cytoplasm or nuclear after DCDC2 was overexpressed or knockdown was measured using western blot. (F) Immunofluorescence staining of β-catenin nuclear translocation in LX-2 cells following DCDC2 was overexpressed. Scale bar, 50 µM. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with Con, # compare with TGF-β1 +VE group (A, B), OE group (C) or sgRNA group (D), *, # P < 0.05.

Article Snippet: After blocking by 5% nonfat milk, the membranes were incubated with the primary antibody against the following targets: DCDC2 (26,978–1-AP, Proteintech, Wuhan, China), α-SMA (ab124964, Abcam, England), Col1α1 (AF6524, Beyotime Biotech, Shanghai, China), β-catenin (GB12015, Servicebio, Wuhan, China), CylcinD1 (26,939–1-AP, Proteintech, Wuhan, China), c-myc (10,828–1-AP, Proteintech, Wuhan, China), Lamin A/C (10,298–1-AP, Proteintech, Wuhan, China), E-cadherin (20,874–1-AP, Proteintech, Wuhan, China), N-cadherin (22,018–1-AP, Proteintech, Wuhan, China), Vimentin (10,366–1-AP, Proteintech, Wuhan, China), Snail1 (13,099–1-AP, Proteintech, Wuhan, China) and GAPDH (60,004-1-Ig, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Activation Assay, Expressing, Western Blot, Plasmid Preparation, Negative Control, Transfection, Knockdown, Immunofluorescence, Staining, Translocation Assay

Journal: Scientific reports

Article Title: DCDC2 inhibits hepatic stellate cell activation and ameliorates CCl 4 -induced liver fibrosis by suppressing Wnt/β-catenin signaling.

doi: 10.1038/s41598-024-59698-w

Figure Lengend Snippet: Figure 4. DCDC2 suppresses HSC proliferation and attenuates EMT-like transitions in vitro. (A,B) The cell cycle phase distribution was measured using flow cytometry analyses after LX-2 cells were transfected with DCDC2 overexpressing lentiviral vector or DCDC2-sgRNA. (C,D) EdU assay was performed to examine the proliferation of LX-2 cells following DCDC2 overexpression or knockdown. (E) The protein expression of E-cadherin, N-cadherin, vimentin, and Snail1 was detected using western blot after LX-2 cells were transfected with DCDC2 overexpressing lentiviral vector or DCDC2-sgRNA. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with VE group or NC group, *P < 0.05.

Article Snippet: After blocking by 5% nonfat milk, the membranes were incubated with the primary antibody against the following targets: DCDC2 (26,978–1-AP, Proteintech, Wuhan, China), α-SMA (ab124964, Abcam, England), Col1α1 (AF6524, Beyotime Biotech, Shanghai, China), β-catenin (GB12015, Servicebio, Wuhan, China), CylcinD1 (26,939–1-AP, Proteintech, Wuhan, China), c-myc (10,828–1-AP, Proteintech, Wuhan, China), Lamin A/C (10,298–1-AP, Proteintech, Wuhan, China), E-cadherin (20,874–1-AP, Proteintech, Wuhan, China), N-cadherin (22,018–1-AP, Proteintech, Wuhan, China), Vimentin (10,366–1-AP, Proteintech, Wuhan, China), Snail1 (13,099–1-AP, Proteintech, Wuhan, China) and GAPDH (60,004-1-Ig, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: In Vitro, Flow Cytometry, Transfection, Plasmid Preparation, EdU Assay, Over Expression, Knockdown, Expressing, Western Blot

Journal: Scientific reports

Article Title: DCDC2 inhibits hepatic stellate cell activation and ameliorates CCl 4 -induced liver fibrosis by suppressing Wnt/β-catenin signaling.

doi: 10.1038/s41598-024-59698-w

Figure Lengend Snippet: Figure 5. Overexpression of DCDC2 improves liver fibrosis in CCl4-induced mouse model. (A) The timeline of the animal experiment. Two weeks following the initial CCl4 injection, a single dose of lentivirus carrying DCDC2 or the control was administered via the tail vein. After 5 weeks of CCl4 treatment, the mice were sacrificed. (B) The protein expression levels of β-catenin, α-SMA, and Col1α1 were detected by western blot after overexpression of DCDC2 in mice. (C) Immunohistochemistry was performed to detect the expression of β-catenin and α-SMA in tissues from mice treated with either control virus or LV-DCDC2. (D) H&E, Sirius red staining and Masson trichrome staining in CCl4 mice following LV-DCDC2 treatment. (E) Serum ALT and AST levels were determined in CCl4 mice after LV-DCDC2 treatment. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with Con group, # compare with CCl4 group, *, # P < 0.05.

Article Snippet: After blocking by 5% nonfat milk, the membranes were incubated with the primary antibody against the following targets: DCDC2 (26,978–1-AP, Proteintech, Wuhan, China), α-SMA (ab124964, Abcam, England), Col1α1 (AF6524, Beyotime Biotech, Shanghai, China), β-catenin (GB12015, Servicebio, Wuhan, China), CylcinD1 (26,939–1-AP, Proteintech, Wuhan, China), c-myc (10,828–1-AP, Proteintech, Wuhan, China), Lamin A/C (10,298–1-AP, Proteintech, Wuhan, China), E-cadherin (20,874–1-AP, Proteintech, Wuhan, China), N-cadherin (22,018–1-AP, Proteintech, Wuhan, China), Vimentin (10,366–1-AP, Proteintech, Wuhan, China), Snail1 (13,099–1-AP, Proteintech, Wuhan, China) and GAPDH (60,004-1-Ig, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Over Expression, Injection, Control, Expressing, Western Blot, Immunohistochemistry, Virus, Staining