Journal: BMC Cancer

Article Title: Nitric-Oxide Synthase trafficking inducer (NOSTRIN) is an emerging negative regulator of colon cancer progression

doi: 10.1186/s12885-022-09670-6

Figure Lengend Snippet: NOSTRIN-induced alteration of transcripts associated with epithelial mesenchymal transition in HCT116 cells

Article Snippet: Anti-SNAI2/SLUG (Cat no. 9585), anti-SMAD2 (Cat no. 5339), anti-STAT3 (Cat no. 4904), anti-Caldesmon-1/CALD1 (Cat no. 12503), anti-Jagged1/JAG1 (Cat no. 2620), anti-Moesin/MSN (Cat no. 3150), anti-Vimentin/VIM (Cat no. 5741), anti-Integrinα5/ITGα5 (Cat no. 4705), anti-E-cadherin/CDH1 (Cat no. 3195), anti-Integrin-linked protein kinase1/ILK1 (Cat no. 3856), anti-CD44 (Cat no. 5640), anti-EpCAM (Cat no. 36746), anti-CD133 (Cat no. 86781) and anti-GAPDH (Cat no. 2118) antibodies were purchased from Cell Signaling Technology, USA and was used at 1:1000 dilution.

Techniques: Binding Assay

Journal: Acta Neuropathologica

Article Title: Oligosarcomas, IDH-mutant are distinct and aggressive

doi: 10.1007/s00401-021-02395-z

Figure Lengend Snippet: Oligosarcomas have a highly distinct proteome with evidence for smooth muscle differentiation. a Volcano plot of significantly differentially regulated proteins between oligosaroma and conventional grade 3 oligodendroglioma. Using the 500 most variable proteins of the dataset for principal component analysis ( b ) and unsupervised hierarchical clustering ( c ) differentiates oligosarcomas from conventional grade 3 oligodendrogliomas. d Heatmap shows upregulation of (smooth) muscle specific proteins in oligosarcomas (left) and immunohistochemistry confirms expression of CALD1 and ACTA2 (SMA) in tumor cells of oligosarcoma (right). Scale bar is 200 µm

Article Snippet: Primary antibodies were diluted as follows: IDH1 R132H (1:2, clone H09 [ ]), NF1 (1:4, clone NFC [ ]), p53 (1:50, Novocastra, Leica, Wetzlar, Germany), SMA (1:500, Cell Marque, Sigma Aldrich, St. Louis, USA), H3K27me3 (1:25, monoclonal antibody, Cell signalling, Danvers, USA), YAP1 (1:100, Cell signalling), Caldesmon (CALD1) (1:100, clone E89, zytomed-systems, Berlin, Germany), GFAP (1:2000, clone GA5, Cell signalling, Danvers, USA), OLIG2 (1:50, clone ERP 2673, Abcam, Cambridge, UK).

Techniques: Immunohistochemistry, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility

doi: 10.1681/ASN.2018070729

Figure Lengend Snippet: Calpain 1, calpain 2, caldesmon-1 and ERK 1/2 are novel TRPC6 binding partners. (A) Table of proteomics results of TRPC6-binding partners from WT human podocytes overexpressing TRPC6-GFP. TRPC6 was seen to bind to TRPC3, TRPC7, and PLCy2, interactions that have been described previously.49,50 Novel interactions with calpain 2 and caldesmon-1 were identified. Podocytes expressing the GFP protein only were used as a control. (B) Interactions reported by proteomics were confirmed in TRPC6 KO cells expressing WT TRPC6-GFP (T6K+WT) by coimmunoprecipitation (TRAP lane). Control agarose beads were used to demonstrate that immunoprecipitation was specific to TRPC6 (control lane). Additional interactions with calpain 1 and ERK 1/2 were also identified. On the basis of the proteomics results, the phosphorylation and/or cleavage states of FAK, talin-1, caldesmon-1, and ERK 1/2 were ascertained through western blotting. (C) T6K had increased FAK phosphorylation at Tyr 397 and decreased ERK phosphorylation compared with control and T6K+WT cells. (D) Cleavage of FAK, talin-1, and caldesmon-1 was decreased in T6K cells compared with controls. For densitometry see Supplemental Figure 3. Con, control; TRAP, GFP-TRAP associated pull-down.

Article Snippet: 25 – 27 Antibody Supplier and Catalog Number TRPC6 Cell Signaling Technology #16716 Caldesmon-1 Cell Signaling Technology #2980 Calpain 1 Cell Signaling Technology #2556 Calpain 2 Cell Signaling Technology #2539S Calpain 1 Abcam #ab28258 Talin-1 Cell Signaling Technology #4021 Phospho-p44/p42 (ERK1/2) Cell Signaling Technology #4370S FAK Cell Signaling Technology #3285S Phospho-FAK (Tyr397) Cell Signaling Technology #8556S Synaptopodin Santa Cruz #sc-515842 WT1 Cell Signaling technology #13580 Podocin Abcam #ab50339 CD2AP Cell Signaling #5478 Nephrin Acris #BP5030 GFP Sigma #11814460001 CD99 Kind gift from Professor George Banting University of Bristol.

Techniques: Binding Assay, Expressing, Immunoprecipitation, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility

doi: 10.1681/ASN.2018070729

Figure Lengend Snippet: Proposed role of the calpain-TRPC6 interaction in podocytes. (A) WT TRPC6 binds to, and acts as a structural scaffold at the membrane for, caldesmon-1 (cald1), ERK 1/2, and calpain. This keeps calpain localized just below the membrane where it can easily cleave its targets talin-1, FAK, and caldesmon-1. (B) In the absence of TRPC6 there is no calcium influx to the cell and calpain is also not localized to the membrane. This means that there is no cleavage of talin-1, FAK, or caldesmon-1. (C) The disease-causing mutant TRPC6 K874* has a truncation at its C terminus. Calpain no longer binds to this form of TRPC6 and is mislocalized. The mutant allows the same calcium influx as WT TRPC6. There is no cleavage of caldesmon-1, talin-1, or FAK. This suggests that the localization of calpain to the membrane is important in its function in the podocyte.

Article Snippet: 25 – 27 Antibody Supplier and Catalog Number TRPC6 Cell Signaling Technology #16716 Caldesmon-1 Cell Signaling Technology #2980 Calpain 1 Cell Signaling Technology #2556 Calpain 2 Cell Signaling Technology #2539S Calpain 1 Abcam #ab28258 Talin-1 Cell Signaling Technology #4021 Phospho-p44/p42 (ERK1/2) Cell Signaling Technology #4370S FAK Cell Signaling Technology #3285S Phospho-FAK (Tyr397) Cell Signaling Technology #8556S Synaptopodin Santa Cruz #sc-515842 WT1 Cell Signaling technology #13580 Podocin Abcam #ab50339 CD2AP Cell Signaling #5478 Nephrin Acris #BP5030 GFP Sigma #11814460001 CD99 Kind gift from Professor George Banting University of Bristol.

Techniques: Mutagenesis

Journal: Journal of the American Society of Nephrology : JASN

Article Title: TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility

doi: 10.1681/ASN.2018070729

Figure Lengend Snippet: Antibodies Used

Article Snippet: 25 – 27 Antibody Supplier and Catalog Number TRPC6 Cell Signaling Technology #16716 Caldesmon-1 Cell Signaling Technology #2980 Calpain 1 Cell Signaling Technology #2556 Calpain 2 Cell Signaling Technology #2539S Calpain 1 Abcam #ab28258 Talin-1 Cell Signaling Technology #4021 Phospho-p44/p42 (ERK1/2) Cell Signaling Technology #4370S FAK Cell Signaling Technology #3285S Phospho-FAK (Tyr397) Cell Signaling Technology #8556S Synaptopodin Santa Cruz #sc-515842 WT1 Cell Signaling technology #13580 Podocin Abcam #ab50339 CD2AP Cell Signaling #5478 Nephrin Acris #BP5030 GFP Sigma #11814460001 CD99 Kind gift from Professor George Banting University of Bristol.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs Prevent Vincristine-Dependent Cancer-Associated Fibroblasts Formation

doi: 10.3390/ijms20081941

Figure Lengend Snippet: Mesenchymal transdifferentiation in HMEC-1 is modulated by vincristine-treated CAF-like cells. HMEC-1 cells were cultured in medium supplemented with CM isolated from coculture of CAF-like cells and colon cancer (LS180—co-CM1 or LoVo—co-CM2) or CAF-like cells maintained in CM colon cancer cells (LS180—CAFs-CM1 or LoVo—CAFs-CM2) and treated, if necessary, with vincristine (+VIN). Then, elongation ratio ( n = 6) ( A ), level of contraction proteins (caldesmon, tropomyosin), vimentin, and α-SMA (Western blot) ( B ), capillary assay ( n = 9) ( C ), and proliferation ability ( D ) were analyzed. In Western blot assay, GAPDH was used as the loading control. The results are provided as means ± SD ( n = 3); * p < 0.05, *** p < 0.005. The blots are representative of three independent experiments.

Article Snippet: In particular experiments, the blots were incubated with primary antibodies recognizing α-SMA, tropomyosin, caldesmon, vimentin (cell signaling), TUBA, TUBB2, TUBB3, TUBB4, GAPDH and IL-6, TGF-β1 or TGF-β2.

Techniques: Cell Culture, Isolation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs Prevent Vincristine-Dependent Cancer-Associated Fibroblasts Formation

doi: 10.3390/ijms20081941

Figure Lengend Snippet: Mesenchymal transdifferentiation in HMEC-1 is not induced by CM from colon cancer cells treated with vincristine. HMEC-1 cells were cultured in medium supplemented with CM isolated from colon cancer cell lines maintained in CM CAFs and treated, if necessary, with vincristine (+VIN). Then, elongation ratio ( n = 6) ( A ), level of contraction proteins (caldesmon, tropomyosin), vimentin, and α-SMA (Western blot) ( B ), and capillary assay ( n = 9) ( C ) were analyzed. In the Western blot assay, GAPDH was used as the loading control. The results are provided as means ± SD ( n = 3). The blots are representative of three independent experiments.

Article Snippet: In particular experiments, the blots were incubated with primary antibodies recognizing α-SMA, tropomyosin, caldesmon, vimentin (cell signaling), TUBA, TUBB2, TUBB3, TUBB4, GAPDH and IL-6, TGF-β1 or TGF-β2.

Techniques: Cell Culture, Isolation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs Prevent Vincristine-Dependent Cancer-Associated Fibroblasts Formation

doi: 10.3390/ijms20081941

Figure Lengend Snippet: NSAIDs inhibit the effect of vincristine-induced EndMT stimulation. Level of TGF-βs (TGF-β1 and TGF-β2) and IL-6 in CM isolated from coculture of CAF-like cells and colon cancer or CAF-like cells maintained in colon cancer cell CM treated, if necessary, with vincristine ( A ) were studied by Western blot. The results are provided as means ± SD ( n = 3); * p < 0.05, *** p < 0.005. The blots are representative of three independent experiments. As the control of loading, the Ponceau Staining was shown. Then, HMEC-1 cells were cultured in medium supplemented with CM isolated from coculture of CAF-like cells and colon cancer or CAF-like cells maintained in colon cancer cell CM and treated, if necessary, with vincristine (+VIN) or NSAIDs (IBU or AsA). Then, elongation ratio ( n = 6) ( B ), capillary assay ( n = 9) ( C ), and level of contraction protein (caldesmon, tropomyosin), vimentin, and α-SMA were analyzed by Western blot ( D ), with GAPDH used as the loading control. The results are provided as means ± SD ( n = 3), ** p < 0.001, *** p < 0.005. The blots are representative of three independent experiments. The values from control cells are marked by dashed lines.

Article Snippet: In particular experiments, the blots were incubated with primary antibodies recognizing α-SMA, tropomyosin, caldesmon, vimentin (cell signaling), TUBA, TUBB2, TUBB3, TUBB4, GAPDH and IL-6, TGF-β1 or TGF-β2.

Techniques: Isolation, Western Blot, Staining, Cell Culture