Journal: The Journal of Cell Biology

Article Title: Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

doi: 10.1083/jcb.200706028

Figure Lengend Snippet: Immunolabeling of primary cilia in undifferentiated hESCs. (A) Characterization of undifferentiated colonies of H1 hESCs grown on matrigel for 5 d in DME:F12 with serum replacement. Undifferentiated cells are identified by nuclear colocalization of anti–OCT-4 (OCT-4, green) and DAPI (dark blue) in the merged image (light blue). More than 97% of cells on average expressed OCT-4 in a nuclear pattern indicating their undifferentiated state. Primary cilia stained with anti-AcTb (tb, red) are indicated by arrows. (B) H1 hESCs grown on matrigel for 5 d in DME:F12 with serum replacement (i.e., unstarved), labeled with anti-AcTb (tb, green) to show primary cilia (arrows). (C) H1 hESCs grown on matrigel for the same period of time in DME:F12 without serum replacement (i.e., starved) for 24 h and labeled with anti-AcTb (tb, green). Primary cilia are indicated by arrows. (D) Characterization of undifferentiated colonies of LRB003 hESC grown on 0.1% gelatin with conditioned medium. Undifferentiated cells are identified by nuclear colocalization of anti–OCT-4 (OCT-4, green) and DAPI (dark blue) in the merged image (light blue). Anti-AcTb (tb, red) marks the primary cilia (arrows) as well as the microtubular network in the cytoplasm. Less than or equal to 95% of the cells were ciliated and positive for OCT-4. Primary cilia are not easily visualized at the low resolution images (as shown in the insets). (E) A primary cilium (tb, red, arrow) in undifferentiated LRB003 hESCs emerges from one of the centrioles (asterisks) marked with anti-centrin (centrin, green). Nuclear localization of anti–OCT-4 (OCT-4, blue) denotes that the cell has not differentiated. The inset shows anti-pericentrin (Pctn, green) marking the centrosome (¤) at the base of the primary cilium (arrow).

Article Snippet: They were then rinsed three times with PBS, incubated in 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min, and blocked with 2% BSA in PBS for 1 h at RT or overnight at 4°C, and primary antibodies (monoclonal anti-AcTb mouse anti–human IgG2b [Sigma-Aldrich]; purified polyclonal anti–γ-tubulin rabbit anti–human IgG [BioLegend]; purified polyclonal anti-zinc finger protein Gli2 rabbit anti–human IgG [Aviva Systems Biology]; monoclonal anti–Tra-1-85 mouse anti–human IgG1 [Millipore]; and PE-conjugated monoclonal anti–stage-specific embryonic antigen 4 mouse anti–human IgG3 [R&D Systems]) were added in 1:300 dilution in blocking buffer for 1 h at RT or overnight at 4°C.

Techniques: Immunolabeling, Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

doi: 10.1083/jcb.200706028

Figure Lengend Snippet: Hh signaling proteins localize to hESC primary cilia. (A) Localization of YFP:Smo (green) to the primary cilium (tb, red, arrow) in LRB003 hESCs. The merged image shows colocalization. Nuclei are stained with DAPI (blue). (B) Immunolocalization of Gli2 (green) to primary cilia (tb, red, arrows) in LRB003 hESCs. The merged image shows colocalization. Nuclei are stained with DAPI (blue). Gli data from H1 and LRB003 cells were obtained with different antibodies. (C) H1 cells grown for 7 d on matrigel in N2/B27-supplemented medium and labeled for primary cilia (tb, green) and anti-SHh (SHh, red). In addition, a z series of a field showing separate SHh labeling (red) located distinctly to the side of the primary cilium (green) is depicted. (D) Similar cells labeled with anti–Tra-1-85 (red) and anti-Gli2 (Gli2, green). Arrows indicate punctuate localization of the Gli2 protein (green dots) and the inset specifically localizes the Gli2 protein (arrowhead) at one end of a primary cilium (tb, red). (E) Anti-SHh (SHh, red) localizes near the base of most primary cilia. 33/71 cells possess primary cilia (46.5%), which is consistent with . 22/33 cells with primary cilia (66%) exhibit SHh near their base (arrows). SHh also localizes to points not associated with the cilia (arrowheads). The asterisk points to the midbody of cells that have recently undergone cytokinesis. These structures are not associated with Hh signaling molecules. (F) Anti-Smo (Smo, red) localizes predominantly to the base of primary cilia. This pattern of Smo expression is similar to that observed at 0 and 1 h of SAG stimulation (see ). Arrows point to primary cilia (tb, green) and arrowheads indicate Smo localization.

Article Snippet: They were then rinsed three times with PBS, incubated in 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min, and blocked with 2% BSA in PBS for 1 h at RT or overnight at 4°C, and primary antibodies (monoclonal anti-AcTb mouse anti–human IgG2b [Sigma-Aldrich]; purified polyclonal anti–γ-tubulin rabbit anti–human IgG [BioLegend]; purified polyclonal anti-zinc finger protein Gli2 rabbit anti–human IgG [Aviva Systems Biology]; monoclonal anti–Tra-1-85 mouse anti–human IgG1 [Millipore]; and PE-conjugated monoclonal anti–stage-specific embryonic antigen 4 mouse anti–human IgG3 [R&D Systems]) were added in 1:300 dilution in blocking buffer for 1 h at RT or overnight at 4°C.

Techniques: Staining, Labeling, Expressing

Journal: The Journal of Cell Biology

Article Title: Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

doi: 10.1083/jcb.200706028

Figure Lengend Snippet: Translocation of Smo and Ptc in and out of the primary cilium after SAG stimulation. (A) Localization of anti-Smo (green) to the primary cilium of LRB003 cells (tb, red, arrows) at 0 h of SAG treatment. (B and C) Localization of anti-Smo to the primary cilium (arrows) at 1 and 4 h, respectively. The insets in A–C show high resolution (shifted overlays) of Smo (green) in the primary cilium (red). The asterisk indicates the base of the cilium. (D) Localization of anti-Ptc (green) to the primary cilium (tb, red, arrows) at 0 h of SAG treatment. (E and F) Localization of anti-Ptc at 1 and 4 h, respectively, to primary cilium (arrows). The insets in D–F show high resolution images (shifted overlays) of Ptc (green) in the primary cilium (red). Nuclei were stained with DAPI.

Article Snippet: They were then rinsed three times with PBS, incubated in 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min, and blocked with 2% BSA in PBS for 1 h at RT or overnight at 4°C, and primary antibodies (monoclonal anti-AcTb mouse anti–human IgG2b [Sigma-Aldrich]; purified polyclonal anti–γ-tubulin rabbit anti–human IgG [BioLegend]; purified polyclonal anti-zinc finger protein Gli2 rabbit anti–human IgG [Aviva Systems Biology]; monoclonal anti–Tra-1-85 mouse anti–human IgG1 [Millipore]; and PE-conjugated monoclonal anti–stage-specific embryonic antigen 4 mouse anti–human IgG3 [R&D Systems]) were added in 1:300 dilution in blocking buffer for 1 h at RT or overnight at 4°C.

Techniques: Translocation Assay, Staining

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: BMP signaling in CCN2/CTGF treated HTM-N cells in vitro . (A, B) Verification of BMP signaling activity in HMT-N cells in vitro . Immunoreactivity of pSmad1/5/8 (green) in HMT-N cells was increased after the treatment with 10 ng/mL BMP-4 (A) and 10 ng/mL BMP-7 (B) . Nuclei were stained with Dapi (blue). n = 3 (C) Western blot analysis of pSmad1/5/8 in the cytoplasmic fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after the treatment with BMP-4 for. (n = 5). GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (D) Western blot analysis of pSmad1/5/8 in the nuclear fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after 1 h with both treatments (n = 5). LaminB1 was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (E) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced after the treatment with CCN2/CTGF ( Bmp-4 : control n = 4, 50 ng/mL CCN2/CTGF n = 3, 100 ng/mL CCN2/CTGF n = 3; Bmp-7 : control n = 5, 50 ng/mL CCN2/CTGF n = 4, 100 ng/mL CCN2/CTGF n = 3). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (F) Real-time RT-PCR analyses of Smad6 , Smad7 , and Id2 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Smad6 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (n = 6). mRNA expression of Smad7 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression of Id2 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (G) Western blot analyses of BMP-7 after the treatment with 5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. Protein synthesis of BMP-7 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 4, 5 ng/mL CCN2/CTGF: n = 3, 25 ng/mL CCN2/CTGF: n = 3, 50 ng/mL CCN2/CTGF: n = 3, 100 ng/mL CCN2/CTGF: n = 3). Mean value of wildtype animals (control) was set to 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (H) Western blot analysis of pSmad1/5/8 after the treatment with 10 ng/mL BMP-4, 60 ng/mL Noggin and 10 ng/mL BMP-4, and 50 ng/mL CCN2/CTGF and 10 ng/mL BMP-4 in HTM-N cells. pSmad1/5/8 protein synthesis was increased after the treatment with BMP-4 (n = 5), compared to untreated control cells and significantly reduced after the treatment with the combination of Noggin and BMP-4 (n = 5) and the combination of CCN2/CTGF and BMP-4 (n = 5), compared to the treatment with BMP-4 only. Mean value of wildtype animals (control) was set to 1. GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. For statistical analysis the One-way ANOVA test was used. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: In Vitro, Activity Assay, Staining, Western Blot, Two Tailed Test, Quantitative RT-PCR, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: TGF-β signaling in CCN2/CTGF treated HTM-N cells in vitro and in βB1-CTGF1 mice in vivo . (A) Real-time RT-PCR analyses of Tgf-b1 , Tgf-b2 and Ccn2/Ctgf in HTM-N cells after the treatment with 5 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h. mRNA expression of Tgf-b1 was significantly increased after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 8, 5 ng/mL CCN2/CTGF: n = 8, 50 ng/mL CCN2/CTGF: n = 6, 100 ng/mL CCN2/CTGF: n = 3). Tgf-b2 mRNA was significantly enhanced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 5 ng/mL CCN2/CTGF: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 5). mRNA expression of Ccn2/Ctgf was significantly increased after the treatment with 5 ng/mL and 50 ng/mL CCN2/CTGF (control: n = 6, 5 ng/mL CCN2/CTGF: n = 6, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 6). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (B) Western blot analysis of TGF-β1 and TGF-β2 in HTM-N cells after the treatment with 5 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h. Protein synthesis of TGF-β1 was significantly increased after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (n = 4). The protein synthesis of TGF-β2 was significantly enhanced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 6, 5 ng/mL CCN2/CTGF: n = 8, 50 ng/mL CCN2/CTGF: n = 6, 100 ng/mL CCN2/CTGF: n = 5). Right panel shows representative Western Blots for the proteins. Mean value of untreated control cells was set at 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (C) Real-time RT-PCR analysis of Tgf-b1 and Tgf-b2 in the anterior eye segment of 2-month-old βB1-CTGF1 mice and wildtype littermates. mRNA expression of Tgf-b2 was significantly increased in βB1-CTGF mice, compared to wildtype littermates ( Tgf-b1 : WT: n = 8, TG: n = 6; Tgf-b2 : WT: n = 19, TG: n = 19). For statistical analysis the Mann-Whitney test was used. mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. (D) Western blot analysis of TGF-β2 in the anterior eye segment of 2-month-old βB1-CTGF mice and wildtype littermates. Protein synthesis of TGF-β2 was significantly increased in βB1-CTGF1 mice, compared to wildtype littermates (TGF-β2: WT: n = 6, TG: n = 6). Right panel shows a representative Western blot. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Mann-Whitney test was used. (E) Immunohistochemical staining of TGF-β2 (red) in the anterior chamber of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of TGF-β2 was increased in the TM of βB1-CTGF mice1, in comparison to wildtype mice. Nuclei were stained with Dapi (blue). n = 5 (F) Western blot analysis of the phosphorylation of Smad2 and Smad3. Protein synthesis of pSmad2 and pSmad3 was significantly increased in βB1-CTGF mice, compared to wildtype littermates (pSMAD2: WT: n = 6, TG: n = 6; pSmad3: WT: n = 7, TG: n = 7). Right panel shows representative Western Blots for both proteins. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Mann-Whitney test was used. (G) Immunohistochemical staining of pSmad2 (red) in the anterior chamber angle of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of pSmad2 is increased in the TM of βB1-CTGF1 mice, in comparison to wildtype mice. Nuclei were stained with Dapi (blue). Data represented as mean ± SD. CB: ciliary body, I: iris, C: cornea, TM: trabecular meshwork. n = 5. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY, Immunohistochemical staining, Staining

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: CCN2/CTGF induced TGF-β signaling activation is mediated via the Erk- and RhoA/ROCK signaling pathway. (A) Real-time RT-PCR analysis of Tgf-b1 , Tgf-b2 and Ccn2/Ctgf in HTM-N cells after the inhibition of the Erk-pathway. After treatment with CCN2/CTGF expression of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf was significantly higher than in DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in expression compared to DMSO treated control cells, but the upregulation was significantly inhibited compared to cells treated with CCN2/CTGF only. Tgf-b2 and Ccn2/Ctgf mRNA expression was significantly reduced after the treatment with the Mek1/2 inhibitor only, compared to DMSO treated control cells (n ≥ 4; # p ≤ .05, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). (B) Western blot analysis of TGF-β1 in HTMN-cells after the inhibition of the Erk-pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β1 is significantly increased compared to DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n ≥ 4, # p ≤ 0.05 to DMSO control; * p ≤ 0.05 to CCN2/CTGF treatment only). Right panel shows a representative Western blot. Mean value of untreated control cells was set at 1. α -Tubulin was used to normalize protein synthesis. (C) Western blot analysis of TGF-β2 in HTMN-cells after the inhibition of the Erk-pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β2 is significantly increased compared to DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 4, # p ≤ 0.05 to DMSO control; * p ≤ 0.05 to CCN2/CTGF treatment only). Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. (D) Real-time RT-PCR analyses of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf in HTM-N cells after the inhibition of the RhoA/ROCK signaling pathway. After treatment with CCN2/CTGF expression of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf was significantly higher than in DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in expression compared to DMSO treated control cells, but the upregulation was significantly inhibited compared to cells treated with CCN2/CTGF only. Tgf-b2 and Ccn2/Ctgf mRNA expression was significantly reduced after the treatment with the Fasudil only, compared to DMSO treated control cells (n ≥ 5; # p ≤ 0.05, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). (E) Western blot analysis of TGF-β1 in HTMN-cells after the inhibition of the RhoA/ROCK signaling pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β1 is significantly increased compared to DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 3, # p ≤ 0.05 to DMSO control; ** p < 0.01 to CCN2/CTGF treatment only). Right panel shows a representative Western blot. Mean value of untreated control cells was set to1. α -Tubulin was used to normalize protein synthesis. (F) Western blot analysis of TGF-β2 in HTMN-cells after the inhibition of the RhoA/ROCK signaling pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β2 was significantly increased compared to DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 7, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. Dotted line indicates the control group treated with the solvent of the inhibitor for the appropriate experiment. For statistical analysis a one-way ANOVA was performed.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: Activation Assay, Quantitative RT-PCR, Inhibition, Expressing, Western Blot, Staining

Journal: American Journal of Cancer Research

Article Title: NDRG2 inhibits pyruvate carboxylase-mediated anaplerosis and combines with glutamine blockade to inhibit the proliferation of glioma cells

doi:

Figure Lengend Snippet: NDRG2 interacts with pyruvate carboxylase and blocks the upregulation of pyruvate carboxylase expression in glutamine-deprived glioma cells. A. Graphic illustration of the tandem affinity and purification procedure for mass spectrometry (TAP-MS). B. PCR amplification of the NDRG2 gene from human astrocyte cDNA. C. Digestion identification of recombinant plasmid S protein-Flag-Streptavidin binding protein tag-NDRG2 (SFB-NDRG2) with restriction enzyme by agarose gel electrophoresis. D. HEK293T cells were transfected with the recombinant plasmid SFB-NDRG2, and Flag tag expression was detected by immunoblotting using an anti-Flag antibody. E. Purified protein complexes for mass spectrometry were detected by immunoblotting using an anti-Flag antibody. F. Subcellular localization of NDRG2 and pyruvate carboxylase in U251 cells. Immunocytochemical staining of U251 cells was performed using NDRG2 or PC antibody, and visualized using FITC- or Cy3-conjugated secondary antibody to show the localization of NDRG2 (green) and PC (yellow). DAPI staining (blue) reveals the cell nuclei. Scale bar, 20 μm. G. Western blot analysis of ectopically expressed Flag-NDRG2 and Myc-PC reciprocally immunoprecipitated by anti-Myc or anti-Flag in HEK293T cells. H. U251 and T98G cells were infected with NDRG2 or mCherry lentivirus for 48 hours, and then cultured in DMEM medium without glutamine for the indicated periods. The protein levels of pyruvate carboxylase and NDRG2 at different times were detected by Western blotting analysis. The relative protein levels of pyruvate carboxylase to β-actin were quantified by densitometry.

Article Snippet: Immunoblotting was performed according to standard procedures with monoclonal rabbit anti-PC antibody (1:2000, Abcam, ab126707), monoclonal mouse anti-NDRG2 antibody (1:1000, Abnova H00057447-M03), monoclonal mouse anti-Flag antibody (1:1000, Sigma, F3165), monoclonal mouse anti-Myc tag antibody (1:1000, Abcam, ab32), monoclonal mouse anti-HA tag antibody (1:1000, Abcam, ab18181), monoclonal mouse anti-IDH1 (R132H) antibody (1:200, Sigma, ASB4200548), monoclonal mouse anti-α-tubulin antibody (1:1000, Boster, M03989-2) and monoclonal rabbit anti-β-actin antibody (1:1000, Boster, BM3873).

Techniques: Expressing, Purification, Mass Spectrometry, Amplification, Recombinant, Plasmid Preparation, Binding Assay, Agarose Gel Electrophoresis, Transfection, FLAG-tag, Western Blot, Staining, Immunoprecipitation, Infection, Cell Culture

Journal: American Journal of Cancer Research

Article Title: NDRG2 inhibits pyruvate carboxylase-mediated anaplerosis and combines with glutamine blockade to inhibit the proliferation of glioma cells

doi:

Figure Lengend Snippet: NDRG2 induces the degradation of pyruvate carboxylase upon glutamine deprivation. A. U251 and T98G cells were infected with NDRG2 or mCherry lentivirus for 48 hours, cultured in DMEM medium without glutamine and treated with 50 μM cycloheximide for the indicated periods. The protein levels of pyruvate carboxylase and NDRG2 at different times were detected by Western blotting analysis. The relative protein levels of pyruvate carboxylase to β-actin were quantified by densitometry. B. U251 and T98G cells infected with NDRG2 or mCherry lentivirus were cultured in DMEM medium without glutamine and treated with 20 μM MG-132 for 18 hours, and Western blotting analysis of the protein levels of pyruvate carboxylase and NDRG2 was performed. C. Western blotting analysis of the products of the in vivo ubiquitination assay in HEK293T cells transfected with the indicated plasmids and cultured in DMEM medium without glutamine was performed.

Article Snippet: Immunoblotting was performed according to standard procedures with monoclonal rabbit anti-PC antibody (1:2000, Abcam, ab126707), monoclonal mouse anti-NDRG2 antibody (1:1000, Abnova H00057447-M03), monoclonal mouse anti-Flag antibody (1:1000, Sigma, F3165), monoclonal mouse anti-Myc tag antibody (1:1000, Abcam, ab32), monoclonal mouse anti-HA tag antibody (1:1000, Abcam, ab18181), monoclonal mouse anti-IDH1 (R132H) antibody (1:200, Sigma, ASB4200548), monoclonal mouse anti-α-tubulin antibody (1:1000, Boster, M03989-2) and monoclonal rabbit anti-β-actin antibody (1:1000, Boster, BM3873).

Techniques: Infection, Cell Culture, Western Blot, In Vivo, Ubiquitin Proteomics, Transfection

Journal: American Journal of Cancer Research

Article Title: NDRG2 inhibits pyruvate carboxylase-mediated anaplerosis and combines with glutamine blockade to inhibit the proliferation of glioma cells

doi:

Figure Lengend Snippet: The expression of NDRG2 had an inverse association with pyruvate carboxylase in IDH1 (R132H)-mutant glioma cells. A. NDRG2 and pyruvate carboxylase immunostaining of tissue microarrays comprising IDH1 wild-type and IDH1 (R132H) glioma tissues with different differentiation states. Scale bar, 200 μm and 50 μm (magnification). B. Correlation analysis of the staining index for the expression of NDRG2 and PC proteins in the IDH1 wild-type glioma patient specimens (n = 27) and the IDH1-R132H mutant glioma patient specimens (n = 27). Pearson’s product-moment correlation coefficients and the P values are also shown. C. NDRG2 and pyruvate carboxylase expression in the IDH1 wild-type and IDH1 (R132H) glioma U87 cells. D. The relative protein levels of pyruvate carboxylase to β-actin were quantified by densitometry. *P<0.05, **P<0.01.

Article Snippet: Immunoblotting was performed according to standard procedures with monoclonal rabbit anti-PC antibody (1:2000, Abcam, ab126707), monoclonal mouse anti-NDRG2 antibody (1:1000, Abnova H00057447-M03), monoclonal mouse anti-Flag antibody (1:1000, Sigma, F3165), monoclonal mouse anti-Myc tag antibody (1:1000, Abcam, ab32), monoclonal mouse anti-HA tag antibody (1:1000, Abcam, ab18181), monoclonal mouse anti-IDH1 (R132H) antibody (1:200, Sigma, ASB4200548), monoclonal mouse anti-α-tubulin antibody (1:1000, Boster, M03989-2) and monoclonal rabbit anti-β-actin antibody (1:1000, Boster, BM3873).

Techniques: Expressing, Mutagenesis, Immunostaining, Staining