Journal: mBio

Article Title: A bacterial family of fatty acid acyltransferases related to the Shigella effector IcsB

doi: 10.1128/mbio.03890-25

Figure Lengend Snippet: A subset of novel k-FATs exhibits yeast toxicity and IcsB-like fatty acid acyltransferase activity. ( A ) Map of the pRS313 Gal1p2 centromeric plasmid used for the galactose-inducible expression of IcsB and its homologs. ( B )The expression of IcsB and seven of its homologs in the presence of galactose inhibits yeast growth compared to the empty vector (EV) control. The glucose plate indicates that there is no growth inhibition in the absence of expression and that similar amounts of each strain were plated. Four dilutions were plated (non-diluted, 1/10, 1/100, and 1/1,000). The graph represents the quantification of growth on three independent replicates of the 1/1,000 dilution. The error bars represent the standard deviation of the mean. A Student’s t-test with unpaired data and a 95% CI was performed (*, P < 0.05; **, P < 0.1, ***, P < 0.01). ( C ) Protein acylation in the membrane protein fraction of yeast expressing IcsB and its homologs according to the in-gel fluorescence acyltransferase assay (upper panels). -Alk IcsB is the IcsB sample prior to the addition of Alk-16. -Alk IcsB, Alk-16 EV, and Alk-16 IcsB were loaded on both gels to serve as controls. This acylation assay was performed using the strains from B that were induced with galactose for six hours in the presence of Alk-16 for the final 4 h. The total protein in these samples were assessed using the TGX stain (bottom panels). ( D ) The expression of IcsB and of its homologs was assessed by immunoblotting with an antibody binding to their 3× FLAG tag and with the GAPDH as a loading control.

Article Snippet: Primary antibodies used were mouse FLAG-tag (Sigma-Aldrich, F3165), mouse Myc-tag (Genescript, A00704), and rabbit GAPDH (Bioss antibodies, BS-8789R) diluted 1/5,000, 1/1,000, and 1/10,000, respectively.

Techniques: Activity Assay, Plasmid Preparation, Expressing, Control, Inhibition, Standard Deviation, Membrane, Fluorescence, Staining, Western Blot, Binding Assay, FLAG-tag

Journal: mBio

Article Title: A bacterial family of fatty acid acyltransferases related to the Shigella effector IcsB

doi: 10.1128/mbio.03890-25

Figure Lengend Snippet: Mutations in the catalytic site of k-FATs abolish their enzymatic activity. ( A ) Yeast growth inhibition tests of catalytic residues mutants H145A (left), D195A (center), and C306A (right) compared to their WT counterpart in IcsB and in toxic homologs. The graph represents the quantification of growth on three independent replicates with the dilution of yeast cultures 1/1,000. The error bars represent the standard deviation from the mean. A Student’s t -test with unpaired data and a 95% CI was performed (*, P < 0.05; **, P < 0.1, ***, P < 0.01). ( B ) In-gel protein acylation of C306A mutants compared to their WT counterparts (upper panels). The total protein in these samples were assessed using the TGX stain (bottom panels). ( C ) The expression of IcsB and toxic homolog C306A mutants was compared to their WT counterparts by immunoblotting with an antibody binding to their 3×FLAG tag and with the GAPDH as a loading control.

Article Snippet: Primary antibodies used were mouse FLAG-tag (Sigma-Aldrich, F3165), mouse Myc-tag (Genescript, A00704), and rabbit GAPDH (Bioss antibodies, BS-8789R) diluted 1/5,000, 1/1,000, and 1/10,000, respectively.

Techniques: Activity Assay, Inhibition, Standard Deviation, Staining, Expressing, Western Blot, Binding Assay, Control

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