Journal: Journal of Cell Communication and Signaling

Article Title: Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

doi: 10.1007/s12079-023-00757-8

Figure Lengend Snippet: Expression kinetics of Ccn5 , Ccn2 , and Col Iα1 in isolated liver cells. ( A ) mRNA expression of Ccn5, Ccn2 , and Col Iα1 in hepatocytes, hepatic stellate cells (HSC), myofibroblasts (MFB) and portal myofibroblasts (pMF) in passage 2 (p2) cultured for the indicated time intervals was determined by RT-qPCR. Ccn5 and Col Iα1 expression is undetectable in hepatocytes, while in contrast Ccn2 mRNA increases during prolonged culture times in hepatocytes. In activated HSC and transdifferentiated MFB Ccn5 mRNA is up-regulated with the activation state of the cells. This up-regulation is even more pronounced than the expression of Ccn2 and parallels the induction of Col Iα1 in the course of HSC to MFB transition. Activated pMF displaying a MFB-like phenotype express large quantities of Ccn5 , Ccn2 , and Col Iα1 mRNAs. ( B ) Protein analysis confirms the absence of CCN5 in hepatocytes and the expression in HSC/MFB and PMF. In this analysis, Vimentin and α-SMA served as markers to demonstrate the mesenchymal phenotype of HSC, MFB and pMF. LCN2 was used as a marker for hepatocytes. Fibronectin and Col Iα1 expression confirmed elevated expression of extracellular matrix proteins during transition of HSC to MFB, while the discrimination between pMF and HSC/MFB was done by probing for Desmin

Article Snippet: The two expression vectors RC204636 and MR203197 encoding full-length human or mouse Myc-DDK-tagged CCN5/WISP2 were obtained from Origene.

Techniques: Expressing, Isolation, Cell Culture, Quantitative RT-PCR, Activation Assay, Marker

Journal: Journal of Cell Communication and Signaling

Article Title: Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

doi: 10.1007/s12079-023-00757-8

Figure Lengend Snippet: Ccn5 expression in the inflammatory/fibrogenic bile duct obstruction model. ( A ) Mice were subjected to bile duct ligation (BDL) for 2 weeks. Hepatic expression of indicated genes was compared to sham-operated control animals by RT-qPCR showing that Col Iα1 , Acta2 , Lcn2 , and Tgfb1 are strongly upregulated during hepatic injury. Similarly, the expression of Ccn1 and Ccn2 in fibrotic liver tissue is increased, while the expression of Ccn3 is comparable in healthy and diseased livers. Interestingly, the expression of Ccn5 is significantly upregulated in diseased livers. Differences between the groups reaching significance are marked by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001). ( B ) Protein analysis confirms the increased expression of α-SMA and Col Iα1 and the elevated expression of CCN5 in livers of animals subjected to BDL. ( C ) Densitometric analysis of Western blot data depicted in ( B )

Article Snippet: The two expression vectors RC204636 and MR203197 encoding full-length human or mouse Myc-DDK-tagged CCN5/WISP2 were obtained from Origene.

Techniques: Expressing, Ligation, Control, Quantitative RT-PCR, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

doi: 10.1007/s12079-023-00757-8

Figure Lengend Snippet: Effects of adenoviral overexpression of CCN5 in portal myofibroblasts. ( A ) Cultured portal myofibroblasts were infected for indicated time intervals with adenoviral expression vectors directing expression of murine CCN5, human CCN5 or Luciferase as a control. Mock infected cells served as a further control. Total RNA was isolated and the expression of mCcn5 , hccn5 , Col Iα1 , and Acta2 determined by RT-qPCR. ( B ) Protein analysis confirms the prominent overexpression of the transgenes (CCN5, Luciferase). Both CCN5 transgenes cause a reduction in Col Iα1 expression after infection for 48 h, 72 h, and 96 h, as evaluated by densitometric analysis of the depicted Western blot results. On the other hand, the effect on α-SMA expression is much less, leading to a lower expression only at 72 h and 96 h. ( C ) Western blot analysis demonstrates that overexpression of CCN5 results in reduced expression of the TGF-β type I receptor (ALK5, TβRI) and TGF-β type II receptor (TβRII). As a mutual consequence of the lowered receptor expression, Smad2 gets less activated from 48 h on, while at 72 h and 96 h after overexpression of CCN5 the genuine BMP or „alternative “ TGF-β1/Smad pathway as assessed by increased Smad1/5/8 phosphorylation is activated. In addition, Jun B is strongly up-regulated in cells overexpressing CCN5. GAPDH expression was used in Western blot analysis to document equal protein loading. Depicted is a representative experiment of an analysis that was done twice. The densitometric analysis of the Western blot is shown in Suppl. Figure 6

Article Snippet: The two expression vectors RC204636 and MR203197 encoding full-length human or mouse Myc-DDK-tagged CCN5/WISP2 were obtained from Origene.

Techniques: Over Expression, Cell Culture, Infection, Expressing, Luciferase, Control, Isolation, Quantitative RT-PCR, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

doi: 10.1007/s12079-023-00757-8

Figure Lengend Snippet: Impact of CCN5 on TGF-β signaling. Portal myofibroblasts were infected with adenoviral vectors expressing either human CCN5 (hCCN5) or the luciferase reporter gene (Luc). After 48 h, the cells were stimulated with the indicated concentrations of recombinant TGF-β1. Protein extracts and supernatants were prepared and tested for expression of Col Iα1, Fibronectin, α-SMA, CHOP, GAPDH ( left panel, cell lysate ), Col Iα1, Fibronectin, hCCN5 ( middle panel, culture supernatant ), and GRP94, GRP78, CCN2, and CCN5 ( right panel, cell lysate ). The expression of β-actin was used to document equal protein loading in each lane. Of note, there was no significant effect on the extracellular matrix components Col Iα1, Fibronectin and the activation marker α-SMA, but a strong TGF-β1 independent up-regulation of the UPR related proteins GRP94, GRP78 and CHOP. The densitometric analysis of the three Western blots is shown in Suppl. Figure 7

Article Snippet: The two expression vectors RC204636 and MR203197 encoding full-length human or mouse Myc-DDK-tagged CCN5/WISP2 were obtained from Origene.

Techniques: Infection, Expressing, Luciferase, Recombinant, Activation Assay, Marker, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

doi: 10.1007/s12079-023-00757-8

Figure Lengend Snippet: CCN5 induces endoplasmic reticulum stress in portal myofibroblasts. ( A ) Portal myofibroblasts were infected with indicated adenoviral constructs for indicated time intervals. Subsequently, total RNA was isolated, cDNA synthesized. The expression of unspliced (u) and spliced (s) Xbp1 expression after infection with adenoviral vectors expressing murine or human CCN5 was analyzed by semi-quantitative PCR. Spliced Xbp1 (Xbp1 (s)) transcripts were found in the presence of overexpressed mCCN5 and hCCN5, while cells that were infected with a control virus (Ad-Luc) or cells that were left uninfected showed no spliced Xbp1 . In this analysis, GAPDH was taken as a loading control. ( B ) Relative mRNA expression of Grp94 , Bip and Chop as assessed by RT-qPCR in samples taken from ( A ). ( C ) Western blot analysis of GRP94, GRP78/BIP, IRE1α, ATF6, peIF2α, eIF2α, ATF4, CHOP, pJNK, JNK2, cytochrome c, cleaved caspase-9, and cleaved caspase-3 in protein samples taken from portal myofibroblasts that were infected with indicated adenoviral vectors for indicated time intervals. Expression of GAPDH was used to demonstrate equal protein loading in each lane. The densitometric analysis of the Western blot is given in Suppl. Figure 8

Article Snippet: The two expression vectors RC204636 and MR203197 encoding full-length human or mouse Myc-DDK-tagged CCN5/WISP2 were obtained from Origene.

Techniques: Infection, Construct, Isolation, Synthesized, Expressing, Real-time Polymerase Chain Reaction, Control, Virus, Quantitative RT-PCR, Western Blot

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 1. Low WISP2 expression predicts reduced survival of AML patients. A, correlation of WISP2 expression with the survival probability of AML patients via the UALCAN cancer database. B, analysis of WISP2 expression in AML patients with different subtypes via the UALCAN cancer database. C, Western blot analysis of WISP2 expression in bone marrow mononuclear cells from six healthy donors and 16 AML patients. D, Co-IP assay to detect the acetylation of WISP2 in bone marrow mononuclear cells from healthy donors and AML patients. E, the protein expression and acetylation level of WISP2 in normal bone marrow mononuclear cells and AML cells. Ac, acetylated lysine; AML, acute myeloid leukemia; Co-IP, co-immunoprecipitation; WISP2, Wnt-1- induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 2. WISP2 overexpression inhibits the proliferation and promotes the apoptosis of AML cells. A and B, WISP2 lentivirus or negative control (NC) was infected into HL-60 cells (A) or Kasumi-1 cells (B) for 24, 48, 72 h, 96 h, and 120 h, and cell viability was determined by CCK8 assay. Data represent the mean ± SD (n = 5 for each group). C and D, apoptosis of HL-60 cells (C) or Kasumi-1 cells (D) was measured by flow cytometry after 72 h of infection with LV-WISP2 or LV-NC. Data represent the mean ± SD of three independent experiments. E and F, FACS analysis of the mitochondrial membrane potential of HL-60 cells (E) or Kasumi-1 cells (F) after 72 h of infection with lentivirus. Data represent the mean ± SD of three independent experiments. G and H, Western blot for WISP2 and apoptosis-related markers in whole cell lysates, as well as cytochrome C in mitochondrial and cytoplasmic fractions of HL-60 cells (G) or Kasumi-1 cells (H). p values in Panels A and B were determined with two-way ANOVA followed by Tukey’s multiple comparisons test, and the p values in Panels C–F were determined with one-way ANOVA followed by Tukey’s test (**p < 0.01, ****p < 0.0001). AML, acute myeloid leukemia; cl cas-3, cleaved caspase-3; cl cas-9, cleaved caspase-9; cl PARP, cleaved PARP; cyt c, cytochrome C; cyto, cytoplasmic; LV, lentivirus; LV-WISP2, lentivirus plasmids containing full-length WISP2; mito, mitochondrial; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Over Expression, Negative Control, Infection, CCK-8 Assay, Cytometry, Membrane, Western Blot

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 3. WISP2 overexpression exerts antileukemic effects in the in vivo model of AML. A, percentage of CD45+ cells in the bone marrow of mice transplanted with HL-60 cells. B, Leishman–Wright–Giemsa staining of mouse bone marrow. Black arrows indicate ring-like differentiated cells. The p value in Panel A was determined with an unpaired t test (****p < 0.0001). AML, acute myeloid leukemia; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Over Expression, In Vivo, Staining

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 4. Pan-HDAC inhibitors induce apoptosis and efficiently prevent WISP2 degradation in AML cells. Two pan-HDAC inhibitors, VPA and TSA, were used to treat AML cells. A and B, HL-60 cells and Kasumi-1 cells were treated with VPA or TSA for 24 h and then subjected to CCK8 assay. Data represent the mean ± SD (n = 5 for each group). C and D, apoptosis of AML cells was measured by flow cytometry after 24 h of VPA or TSA treatment. Data represent the mean ± SD of three independent experiments. E and F, quantitative analysis of the percentage of apoptosis. G and H, Western blot for WISP2 in VPA- or TSA-treated AML cells. I and J, the acetylation of WISP2 in VPA- or TSA-treated AML cells was assessed by co-IP assay. K and L, AML cells were treated with VPA or TSA for 12 h and treated with CHX (25 μg/ml) for the indicated times, followed by Western blot analysis of WISP2 expression. p values were determined with one-way ANOVA followed by Dunnett’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Ac, acetylated lysine; AML, acute myeloid leukemia; CHX, cycloheximide; co-IP, co-immunoprecipitation; HDAC, histone deacetylase; TSA, trichostatin A; VPA, valproic acid; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: CCK-8 Assay, Cytometry, Western Blot, Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Histone Deacetylase Assay

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 5. HDAC3 interacts with and deacetylates WISP2 in AML cells. A and B, co-IP was performed with anti-WISP2 antibody and IgG as a control, followed by immunoblotting with anti-HDAC1, anti-HDAC3, anti-HDAC6, and anti-HDAC7 antibodies in AML cells. C, the correlation between HDAC3 or HDAC1 and WISP2 expression levels from 151 AML samples analyzed by StarBase. D, Western blot analysis of HDAC3 expression in bone marrow mononuclear cells from healthy donors and AML patients. E and F, the protein and acetylation levels of WISP2 in RGFP966-treated AML cells. G and H, the protein and acetylation levels of WISP2 and the protein expression of HDAC3 in AML cells infected with HDAC3 lentivirus. Ac, acetylated lysine; AML, acute myeloid leukemia; co-IP, co-immunoprecipitation; HDAC, histone deacetylase; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Co-Immunoprecipitation Assay, Control, Western Blot, Expressing, Infection, Immunoprecipitation, Histone Deacetylase Assay

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 6. The lysine K6 residue is essential for WISP2 acetylation. A, prediction of acetylation on internal lysine residues indicated two potential acetylated lysine (K) residues, K6 and K20, within WISP2. B, HEK293 cells were transfected with wildtype WISP2 (wt-WISP2-flag), mutant WISP2_K6R (WISP2_K6R-flag), or mutant WISP2_K20R (WISP2_K20R-flag) for 48 h, and the transfection efficiency was determined by immunoblotting with an anti-Flag antibody. C and D, HEK293 cells were transfected with wt-WISP2-flag, WISP2_K6R-flag, or WISP2_K20R-flag for 48 h and then treated with VPA or RGFP966 for 24 h. Whole-cell lysates were subjected to detection with anti-acetylated-lysine antibody. Ac, acetylated lysine; VPA, valproic acid; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Residue, Transfection, Mutagenesis, Western Blot

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 7. Acetylation of WISP2 prevents NEDD4-mediated ubiquitination. A, HL-60 cells were treated with or without RGFP966 for 24 h, and MG132 was added 6 h before harvesting cells. Proteins were co-IP with anti-WISP2 antibody, followed by immunoblotting with anti-ubiquitin or anti-WISP2 antibodies. B, HDAC3 lentivirus was infected into HL-60 cells for 72 h, and MG132 was added 6 h before harvesting cells. The ubiquitination of WISP2 was detected by co-IP with an anti-WISP2 antibody, followed by immunoblotting with the indicated antibodies. C, HL-60 cells were infected with HDAC3 lentivirus. After 72 h, the cells were treated with MG132 for 6 h. Total lysates were analyzed by Western blotting using an anti-WISP2 antibody. D, prediction of Ubibrowser indicated that NEDD4 was the top E3 ubiquitin ligase that probably targets WISP2. E and F, endogenous interaction between WISP2 and NEDD4 in HL-60 cells and Kasumi-1 cells was examined by co-IP assay. G and H, the protein levels of NEDD4 and WISP2 were confirmed by Western blotting in NEDD4- overexpressing AML cells. I, co-IP assay to assess WISP2 ubiquitination in HL-60 cells infected with NEDD4 lentivirus. J, co-IP assay to assess WISP2 ubiquitination in HL-60 cells transfected with NEDD4 overexpression plasmid and wt-WISP2-flag or WISP2_K6R-flag. K, HL-60 cells were infected with LV-oeHDAC3 and LV-shNEDD4 as indicated. Western blot analysis was performed to determine WISP2 expression. L, schematic diagram for the antileukemic effect of WISP2: HDAC3-induced deacetylation disrupts WISP2 stability by augmenting ubiquitination mediated by NEDD4, which contributes to the in- crease in tumor cell proliferation and the suppression of apoptosis. The blockade of histone deacetylation by HDAC inhibitors restores WISP2 expression in AML cells, thereby allowing WISP2 to exert its antileukemia effect. AML, acute myeloid leukemia; co-IP, coimmunoprecipitation; LV, lentivirus; ub, ubiquitin; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Western Blot, Infection, Transfection, Over Expression, Plasmid Preparation, Expressing

Journal: Journal of Cell Communication and Signaling

Article Title: Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

doi: 10.1007/s12079-023-00757-8

Figure Lengend Snippet: Effects of adenoviral overexpression of CCN5 in portal myofibroblasts. ( A ) Cultured portal myofibroblasts were infected for indicated time intervals with adenoviral expression vectors directing expression of murine CCN5, human CCN5 or Luciferase as a control. Mock infected cells served as a further control. Total RNA was isolated and the expression of mCcn5 , hccn5 , Col Iα1 , and Acta2 determined by RT-qPCR. ( B ) Protein analysis confirms the prominent overexpression of the transgenes (CCN5, Luciferase). Both CCN5 transgenes cause a reduction in Col Iα1 expression after infection for 48 h, 72 h, and 96 h, as evaluated by densitometric analysis of the depicted Western blot results. On the other hand, the effect on α-SMA expression is much less, leading to a lower expression only at 72 h and 96 h. ( C ) Western blot analysis demonstrates that overexpression of CCN5 results in reduced expression of the TGF-β type I receptor (ALK5, TβRI) and TGF-β type II receptor (TβRII). As a mutual consequence of the lowered receptor expression, Smad2 gets less activated from 48 h on, while at 72 h and 96 h after overexpression of CCN5 the genuine BMP or „alternative “ TGF-β1/Smad pathway as assessed by increased Smad1/5/8 phosphorylation is activated. In addition, Jun B is strongly up-regulated in cells overexpressing CCN5. GAPDH expression was used in Western blot analysis to document equal protein loading. Depicted is a representative experiment of an analysis that was done twice. The densitometric analysis of the Western blot is shown in Suppl. Figure 6

Article Snippet: The vectors used for transfection were Myc-DDK-tagged pCMV6-Entry-mCCN5 (#MR203197, Origene, Herford, Germany) and Myc-DDK-tagged pCMV6-Entry hCCN5 (#RC204636, Origene).

Techniques: Over Expression, Cell Culture, Infection, Expressing, Luciferase, Control, Isolation, Quantitative RT-PCR, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

doi: 10.1007/s12079-023-00757-8

Figure Lengend Snippet: Impact of CCN5 on TGF-β signaling. Portal myofibroblasts were infected with adenoviral vectors expressing either human CCN5 (hCCN5) or the luciferase reporter gene (Luc). After 48 h, the cells were stimulated with the indicated concentrations of recombinant TGF-β1. Protein extracts and supernatants were prepared and tested for expression of Col Iα1, Fibronectin, α-SMA, CHOP, GAPDH ( left panel, cell lysate ), Col Iα1, Fibronectin, hCCN5 ( middle panel, culture supernatant ), and GRP94, GRP78, CCN2, and CCN5 ( right panel, cell lysate ). The expression of β-actin was used to document equal protein loading in each lane. Of note, there was no significant effect on the extracellular matrix components Col Iα1, Fibronectin and the activation marker α-SMA, but a strong TGF-β1 independent up-regulation of the UPR related proteins GRP94, GRP78 and CHOP. The densitometric analysis of the three Western blots is shown in Suppl. Figure 7

Article Snippet: The vectors used for transfection were Myc-DDK-tagged pCMV6-Entry-mCCN5 (#MR203197, Origene, Herford, Germany) and Myc-DDK-tagged pCMV6-Entry hCCN5 (#RC204636, Origene).

Techniques: Infection, Expressing, Luciferase, Recombinant, Activation Assay, Marker, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

doi: 10.1007/s12079-023-00757-8

Figure Lengend Snippet: CCN5 induces endoplasmic reticulum stress in portal myofibroblasts. ( A ) Portal myofibroblasts were infected with indicated adenoviral constructs for indicated time intervals. Subsequently, total RNA was isolated, cDNA synthesized. The expression of unspliced (u) and spliced (s) Xbp1 expression after infection with adenoviral vectors expressing murine or human CCN5 was analyzed by semi-quantitative PCR. Spliced Xbp1 (Xbp1 (s)) transcripts were found in the presence of overexpressed mCCN5 and hCCN5, while cells that were infected with a control virus (Ad-Luc) or cells that were left uninfected showed no spliced Xbp1 . In this analysis, GAPDH was taken as a loading control. ( B ) Relative mRNA expression of Grp94 , Bip and Chop as assessed by RT-qPCR in samples taken from ( A ). ( C ) Western blot analysis of GRP94, GRP78/BIP, IRE1α, ATF6, peIF2α, eIF2α, ATF4, CHOP, pJNK, JNK2, cytochrome c, cleaved caspase-9, and cleaved caspase-3 in protein samples taken from portal myofibroblasts that were infected with indicated adenoviral vectors for indicated time intervals. Expression of GAPDH was used to demonstrate equal protein loading in each lane. The densitometric analysis of the Western blot is given in Suppl. Figure 8

Article Snippet: The vectors used for transfection were Myc-DDK-tagged pCMV6-Entry-mCCN5 (#MR203197, Origene, Herford, Germany) and Myc-DDK-tagged pCMV6-Entry hCCN5 (#RC204636, Origene).

Techniques: Infection, Construct, Isolation, Synthesized, Expressing, Real-time Polymerase Chain Reaction, Control, Virus, Quantitative RT-PCR, Western Blot