Journal: Cell Death & Disease

Article Title: TDAG51 induces renal interstitial fibrosis through modulation of TGF-β receptor 1 in chronic kidney disease

doi: 10.1038/s41419-021-04197-3

Figure Lengend Snippet: A Rat renal fibroblasts were treated with TM and the IRE1 endonuclease inhibitor 4μ8c. TM-induced splicing of XBP1, which was prevented by 4μ8c treatment. B Renal fibroblasts were treated with TGF-β1 (Tβ) and ascorbic acid (AA2P; AA) to induce XBP1 splicing. Individually, TGF-β1 and AA2P increased XBP1 splicing, but the effect was increased with combined treatment. Co-treatment with 4μ8c was able to prevent TGF-β1-mediated XBP1 splicing. C Renal fibroblasts treated with TGF-β1 and AA2P show increased collagen deposition, which was inhibited with 4μ8c co-treatment. Bar = 100 μm. * P < 0.05 vs veh; *** P < 0.001 vs veh; #, P < 0.05 vs AA2P + TGF-β1. D Western blotting demonstrates that expression of TGF-β1 receptor (TGFβ-R1) is significantly reduced in kidneys from WT mice with CKD. Kidneys from TDKO mice with or without CKD do not express TGFβ-R1. * P < 0.05 vs sham; #, P < 0.05 vs WT. E Renal fibroblasts were treated with TGF-β1 and AA2P with or without SB431542 (SB), a TGFβ-R1 inhibitor. While TGF-β1 and AA2P increased mRNA levels of spliced XBP1, co-treatment with SB431542 inhibited XBP1 splicing. * P < 0.05 vs veh; #, P < 0.05 vs Tβ + AA. F HK-2 cells were treated with thapsigargin for varying time points (0, 18, 24, 48 h) and subsequently underwent western blotting. Thapsigargin induced TDAG51 expression at 18 and 24 h, but not 48 h. TGFβ-R1 was increased after 18 h of TG, but the expression was reduced at 24 and 48 h. * P < 0.05 vs 0 h.

Article Snippet: Antibodies to TDAG51 (sc-23866; Santa Cruz Biotechnology; Santa Cruz, CA, USA) and CHOP (sc-7351; Santa Cruz) were diluted 1:200, KDEL (SPA-827; Stressgen; Burlington, Canada) was diluted 1:1000 (used against the KDEL amino acid sequence of GRP78), TGFβ-R1 (MAB587; R&D systems; Oakville, Canada) was diluted 1:500, and β-actin (66009-1; ProteinTech; Rosemont, IL, USA) was diluted 1:5000.

Techniques: Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: TDAG51 induces renal interstitial fibrosis through modulation of TGF-β receptor 1 in chronic kidney disease

doi: 10.1038/s41419-021-04197-3

Figure Lengend Snippet: The induction of CKD, caused by reduced renal mass, increased angiotensin II and aldosterone, and increased salt consumption, triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). This amplifies TDAG51 expression in the kidney. The elevated levels of TDAG51 triggers a pro-apoptotic signaling pathway and a pro-fibrotic signaling pathway. In the pro-apoptotic pathway, TDAG51 induces expression of CHOP, which activates pro-apoptotic genes and, eventually, programmed cell death. In the pro-fibrotic pathway, TDAG51 increases expression of TGF-β receptor 1, which leads to the splicing of XBP1. Spliced XBP1 leads to the activation of pro-fibrotic genes, and the development of renal interstitial fibrosis.

Article Snippet: Antibodies to TDAG51 (sc-23866; Santa Cruz Biotechnology; Santa Cruz, CA, USA) and CHOP (sc-7351; Santa Cruz) were diluted 1:200, KDEL (SPA-827; Stressgen; Burlington, Canada) was diluted 1:1000 (used against the KDEL amino acid sequence of GRP78), TGFβ-R1 (MAB587; R&D systems; Oakville, Canada) was diluted 1:500, and β-actin (66009-1; ProteinTech; Rosemont, IL, USA) was diluted 1:5000.

Techniques: Activation Assay, Expressing

Journal: International journal of cancer

Article Title: Antitumor activity of ALK1 in pancreatic carcinoma cells.

doi: 10.1002/ijc.22393

Figure Lengend Snippet: FIGURE 1 – ALK1 is expressed and functional in pancreatic carcinoma cells. (a) RT-PCR detection of ALK1 in established pancreatic carci- noma cell lines. Total RNA was isolated from subconfluent cultures of the indicated cell lines and subjected to reverse transcription. Subse- quently, a cDNA amount corresponding to 200 ng of input RNA was amplified in a standard end-point RT-PCR with primers encompassing the entire coding region of the human ALK1 gene. Note the presence of a product corresponding to the full-length receptor (1516 bp) and a promi- nent band of 950 bp. Human umbilical vein endothelial cells (HUVEC) served as positive control. Amplification of b-actin was carried out in parallel to control for the integrity of the cDNA preparations. (b) ALK1 protein is expressed in PANC-1 cells. Endogenous ALK1 protein was immunoprecipitated from 1 mg of PANC-1 total cellular lysate using goat polyclonal antibody (pab) directed against the extracellular domain of recombinant (r) human ALK1. Fractionated immunoprecipitates were blotted and probed with a murine monoclonal antibody (mab) to the same antigen followed by chemoluminescent antigen detection. The arrow indicates the specific band at 65 kDa, which disappeared upon blocking of ALK1 mab binding in the presence of a 10-fold molar excess of rALK1-Fc fusion protein; ns, nonspecific band. (c) Staining for ALK1 (mab) in pancreatic carcinoma (Ca) and peritumoral normal (NP) tissues. Control panels show the corresponding isotype (IgG1) negative controls. (d) TGF-b induces serine phosphorylation of Smad1 and Smad5 in PANC-1 cells. PANC-1 cells were transiently transfected with FLAG-tagged versions of either Smad1 (upper panel), Smad5 (middle panel) or Smad2 as control (lower panel) and 24 hr later stimulated with TGF-b for 1 hr. Cellular lysates (200 lg) were subjected to IP with anti-FLAG antibody and immunoprecipitates were electrophoresed and immunoblotted sequentially for antiphosphoserine (p-Serine) and Smad1, Smad5 or Smad2. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Article Snippet: Antibodies to ALK5 (V-22), Smad1 (E-4), Smad5 (D-20) and GADD45b (N-20) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany), to ALK1 (mouse mab clone 117720 and goat pab) from R&D Systems, to phospho-p38, p38, phospho-MKK3/6, MKK3, phospho-Smad2 and phosphoSmad1/5/8 from Cell Signaling Technology (Heidelberg, Germany), to phospho-Smad3 from Biosource (Solingen, Germany), to Smad2 and phospho-serine from Zymed (Berlin, Germany), to b-actin and FLAG (M2) from Sigma (Deisenhofen, Germany), to hemagglutinin (HA, C125A) from Roche Diagnostics (Mannheim, Germany) or Miltenyi Biotech (Bergisch-Gladbach, Germany), to Ki67 (mouse mab clone MIB-1) from Dianova (Hamburg, Germany) and to CD31 (rat mab clone MEC13.3) from BD Pharmingen (Hamburg).

Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Reverse Transcription, Positive Control, Control, Immunoprecipitation, Recombinant, Blocking Assay, Binding Assay, Staining, Phospho-proteomics, Transfection

Journal: International journal of cancer

Article Title: Antitumor activity of ALK1 in pancreatic carcinoma cells.

doi: 10.1002/ijc.22393

Figure Lengend Snippet: FIGURE 2 – Effect of ectopic expression of caALK1 on Smad activation and Smad-mediated transcription in pancreatic cancer cells. (a) HA- tagged ALK1 proteins from 100 lg of total cell lysate of individual clones (#4 and #10) and polyclonal (pool) PANC-1 cultures were adsorbed to magnetic bead-immobilized anti-HA ab (IP) followed by immunoblotting (IB) for ALK1. Vec, empty vector-transduced control (pool); pt, pa- rental. Cellular lysates (20 lg/lane) from the same cells were immunoblotted for phospho-Smad1/5 (p-Smad1/5). (b) and (c) Vector and caALK1 transduced PANC-1 cells were stimulated for the times indicated with TGF-b1 (5 ng/ml) and processed for immunoblotting (20 lg cell lysate per lane in PAGE) to detect phosphorylated forms of Smad2 (p-Smad2) and Smad3 (p-Smad3). (d) Effect of caALK1 on the TGF-b/Smad-re- sponsive plasmid p6SBE-luc. Cells were cotransfected with p6SBE-luc (25 ng/well) and pRL-TK (25 ng/well) along with the indicated expres- sion vectors and/or empty vector (pcDNA3). Numbers below the graph indicate the transfected DNA amounts (in ng). Following transfection and stimulation with TGF-b1 (5 ng/ml) or BMP-2 (50 ng/ml) for 24 hr, cells were subjected to luciferase assay. The assay was repeated 3 times yielding very similar results. A representative experiment is shown. Each value represents the normalized mean of 6-fold determinations; bars, mean 6 s.d.

Article Snippet: Antibodies to ALK5 (V-22), Smad1 (E-4), Smad5 (D-20) and GADD45b (N-20) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany), to ALK1 (mouse mab clone 117720 and goat pab) from R&D Systems, to phospho-p38, p38, phospho-MKK3/6, MKK3, phospho-Smad2 and phosphoSmad1/5/8 from Cell Signaling Technology (Heidelberg, Germany), to phospho-Smad3 from Biosource (Solingen, Germany), to Smad2 and phospho-serine from Zymed (Berlin, Germany), to b-actin and FLAG (M2) from Sigma (Deisenhofen, Germany), to hemagglutinin (HA, C125A) from Roche Diagnostics (Mannheim, Germany) or Miltenyi Biotech (Bergisch-Gladbach, Germany), to Ki67 (mouse mab clone MIB-1) from Dianova (Hamburg, Germany) and to CD31 (rat mab clone MEC13.3) from BD Pharmingen (Hamburg).

Techniques: Expressing, Activation Assay, Clone Assay, Western Blot, Plasmid Preparation, Control, Transfection, Luciferase

Journal: International journal of cancer

Article Title: Antitumor activity of ALK1 in pancreatic carcinoma cells.

doi: 10.1002/ijc.22393

Figure Lengend Snippet: FIGURE 3 – Effect of caALK1 on basal and TGF-b1-inhibited proliferation in pancreatic cancer cells. (a) Exponentially growing parental PANC-1 cells and various stably transduced derivatives thereof, as indicated in the legend, were labeled with [3H]thymidine for 4 hr and har- vested by vacuum aspiration on glass fiber filters. The incorporated radioactivity was counted and normalized to the total cell number in each well. Data are given in % relative (Rel.) to control (5 parental) cells set arbitrarily at 100. PANC-1 cells stably expressing caALK5 from TJ-neo served as control. Immunoblots below the graph show expression of p21WAF1, the expression level of which should be assessed relative to b- actin as loading control (10 lg of protein were loaded for parental and vector control cells, 20 lg for caALK1-transduced cells). (b) PANC-1 cells stably expressing caALK1 were stimulated for 24 hr with the indicated concentrations of TGF-b1 in normal growth medium. During the last 4 hr cells were labeled with [3H]thymidine. The radioactivity incorporated into DNA was determined for each PANC-1 derivative and plot- ted against the TGF-b concentrations relative to that of the respective unstimulated control set arbitrarily at 100. Growth inhibition by TGF-b1 in pancreatic cells is ALK5-dependent as shown by a clone stably overexpressing kinase-deficient ALK5 (kdALK5). Data in (a) and (b) were derived from 6 wells processed in parallel and are the mean 6 s.d., n 5 3. (c) Cell cycle profile as determined by flow cytometry of untreated and TGF-b-treated COLO-357 cells ectopically expressing empty vector or caALK1. (d) ALK1 expression in COLO-357 clones from (c) detected by combining IP and IB as described for PANC-1 in Figure 2a.

Article Snippet: Antibodies to ALK5 (V-22), Smad1 (E-4), Smad5 (D-20) and GADD45b (N-20) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany), to ALK1 (mouse mab clone 117720 and goat pab) from R&D Systems, to phospho-p38, p38, phospho-MKK3/6, MKK3, phospho-Smad2 and phosphoSmad1/5/8 from Cell Signaling Technology (Heidelberg, Germany), to phospho-Smad3 from Biosource (Solingen, Germany), to Smad2 and phospho-serine from Zymed (Berlin, Germany), to b-actin and FLAG (M2) from Sigma (Deisenhofen, Germany), to hemagglutinin (HA, C125A) from Roche Diagnostics (Mannheim, Germany) or Miltenyi Biotech (Bergisch-Gladbach, Germany), to Ki67 (mouse mab clone MIB-1) from Dianova (Hamburg, Germany) and to CD31 (rat mab clone MEC13.3) from BD Pharmingen (Hamburg).

Techniques: Stable Transfection, Labeling, Radioactivity, Control, Expressing, Western Blot, Plasmid Preparation, Inhibition, Derivative Assay, Cytometry, Clone Assay