Journal: Clinical and Experimental Medicine

Article Title: CD59 silencing enhances doxorubicin sensitivity in DLBCL cells and is associated with poor prognosis in patients treated with CHOP regimen

doi: 10.1007/s10238-026-02134-2

Figure Lengend Snippet: Validation of CD59 knock-down in DLBCL cell lines. ( A ) Representative immunoblots showing CD59 protein levels in SU-DHL-4 and U2932 cell lines transfected with three distinct CD59-shRNA constructs or control vectors. β-actin served as the loading control. ( B and C ) Quantitative analysis of relative CD59 mRNA (by qRT-PCR) and protein (by densitometry) levels in SU-DHL-4 (B) and U2932 (C) cells. Data are presented as mean ± SD and normalized to the blank control group.** p < 0.01; *** p < 0.001

Article Snippet: Mouse anti-human β-actin monoclonal antibody (100166-MM10) was obtained from Sino Biological, and the chemiluminescent substrate SuperSignalTM West Pico PLUS ( ECL34580 ) from Thermo Fisher.

Techniques: Biomarker Discovery, Knockdown, Western Blot, Transfection, shRNA, Construct, Control, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Histological analysis of sleep and circadian brain circuitry in cranial radiation-induced hypersomnolence (C-RIH) mouse model

doi: 10.1038/s41598-022-15074-0

Figure Lengend Snippet: In vitro radiosensitivity of astrocytic cell lines based on region of isolation and treatment timing. ( A ) Photomicrographs of SCN2.2 and CTXTNA2 cells, two astrocytic cell lines with similar morphology. B. Quantification of trypan blue postitive cells to determine cell viability 1, 6 and 24 h after 8 Gy radiation, there were significant main effects for time (F(1,18) = 96.834, p < 0.001), cell type (F(1,18) = 235.937, p < 0.001), and an interaction between time and cell type (F(2,17) = 151.927, < 0.001). Posthoc tests (tukey) were significant if p < 0.05. SCN2.2 cells (green bars) survived at significantly higher level 1 ( p < 0.001) and 6 h ( p = 0.024) after radiation when compared to the cortical astrocytes (purple bars). ( C ) Clonogenic assay to quantify survival fractions from 0 to 8 Gy in SCN and cortical cells. Again SCN2.2 cells (green line) had better overall survival after radiation when compared to CTXTNA2 cells (purple line). There were significant main effects of both cell line (F(1,24) = 33.228, p < 0.001) and radiation dose (F(5,24) = 402.558, p < 0.001), and an interaction between cell line and dose (F(5,24) = 6.172, p = 0.001). The dotted black line across the graph shows the dose modifying fractors (DMF 10 ) levels for both cells lines which was also significantly different between the two cell lines. ( D ) The chronotherapeutic experimental timeline, 1.0 × 10 6 cells were plated on day 0. Cells were then serum shocked with a 50% horse serum solution for 2 h at circadian time(CT) 8 (01:00, Day 2) or 20 (13:00, Day 1) on the subsequent days. All cells were irradiated on Day 2 at 11:00 and then colonies stained and counted after Day 5. ( E ) Clonogenic assay of SCN2.2 cells irradiated at CT8 (dotted line) and CT20 (solid line). Astrocytes irradiated at CT8 were significantly more sensitive to radiotherapy. There were significant main effects of time (F(1,24) = 7.041, p = 0.014) and dose (F(5,24) = 26.995, p < 0.001) but no interaction (F(5,24) = 1.081, p = 0.396). ( F ) Clonogenic assayof CTXTNA2 cells irradiated at CT8 (dotted line) and CT20 (solid line). Again, astrocytes irradiated at CT8 were significantly more sensitive to radiotherapy. There were significant main effect of time (F(1,24) = 14.099, p = 0.001) and dose (F(5,24) = 135.997, p < 0.001) but no interaction (F(5,24) = 1.349, p = 0.278). ( G ) ATM western analysis of both cell lines when samples were collected at two timepoint post-synchronization. Western blot of ATM (blue bands) and control β-actin (teal bands) in both cell lines show higher presence of ATM after serum shock entrainment (Left panel). A graph of the ATM/β-actin relative ratio (Right panel), shows that CTXTNA cells express more of the protein than SCN2.2 cells. Four replicates of each cell line at the 3 timepoints were compared, there were significant main effects of time (F(2,21) = 83.154, p < 0.001) and cell type (F(1,21) = 8.312, p = 0.009) and an interaction (F(2,21) = 5.230, p = 0.014). Posthoc tests (tukey) were significant if p < 0.05. Both cell lines had more protien during CT08 when compared to CT20 but only CTXTNA cells had significant differences between the two ( p = 0.007). Significance was defined as p < 0.05 and indicated by * or a, b, and c with different letters indicating significant differences.

Article Snippet: Membranes were stained using primary antibodies for ATM (1:1000; ab81292, abcam, MA) and β-actin (1:5000; ab8226, abcam, MA) for 24 h at 4°C, followed by 1 h at room temperature in secondary antibodies (1:5000) for IRDye 800CW Goat anti-Mouse (926-32210; Li-Cor Biotechnology, NE) and IRDye 680CW Goat anti-Rabbit (926-68071; Li-Cor Biotechnology, NE).

Techniques: In Vitro, Isolation, IF-P, Clonogenic Assay, Irradiation, Staining, Western Blot, Control

Journal: Nature Communications

Article Title: A reversible state of hypometabolism in a human cellular model of sporadic Parkinson’s disease

doi: 10.1038/s41467-023-42862-7

Figure Lengend Snippet: a Quantification of total mitochondrial mass and functional mitochondria in hNPCs or b DAns. Total mitochondrial mass was visualized using immunostainings with an antibody against the ATP synthase (ATP5F1A). Functional mitochondria with an active membrane potential were visualized using a MitoTracker probe (200 nM for 20 min). Images are exemplarily shown for hNPCs of clone i1E4-R1-003 (Ctrl), and iR66-R1-007 (sPD); for DAns of clone i1E4-R1-003 (mitoTracker—Ctrl), iR66-R1-007 (mitoTracker—sPD), i1JF-R1-018 (ATP5F1A—Ctrl), and iJ2C-R1-015 (ATP5F1A—sPD). Scale bar = 20 μm. c Violin plots highlight some morphological characteristics of mitochondria in hNPCs or d DAns: number of mitochondria (count), mitochondrial area, skeleton length, number of branch points, and mean intensity. On average, 112 (hNPCs—ATP5F1A), 160 (hNPCs—mitoTracker), 370 (DAns—ATP5F1A), 360 (DAns—mitoTracker) cells per clone were analyzed. e To assess alterations in the mitochondrial fusion machinery, expression of the mitofusions MFN1 , MFN2 , and OPA1 was quantified in hNPCs by RT-qPCR. Cells were cultivated on the energy substrates used in the Seahorse XF analysis (25 mM glucose or 5 mM pyruvate). f To assess alterations in the mitochondrial fission machinery, expression of DRP1 and its phosphorylation on Ser 616 was quantified in hNPCs by western blot. Protein levels were normalized to ACTB. g Western blots are exemplary shown for some hNPC clones. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value or to the most extreme data point which is no more than 1.5 times the interquartile range ( c , d ). Each dot represents one patient. n = 5 Ctrl and 7 sPD patient-derived cell clones, in triplicates. p- values were determined by linear mixed effects model ( c , d ); one-way ANOVA with Sidak’s Post hoc test ( p -values are provided together with the source data) ( e , f ). * p < 0.05; ** p < 0.01; *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies NDUFB8 (459210, Novex; 1:500), Complex II- Subunit 30 (SDHB) (459230, Thermo Fisher Scientific; 1:500), UQCRC2 (ab14745, Abcam; 1:2500), MT-CO2 (ab110258, Abcam; 1:1000), ATP5F1A (ab14748, Abcam; 1:4000), DRP1 (5391, Cell Signaling; 1:1000), DRP1 phospho-Ser616 (4494, Cell Signaling; 1:1000), TUBA (GTX628802, Genetex; 1:20,000), ACTB (ABO145-200, OriGene; 1:2000), OGDHL (17110-1-AP, Proteintech, 1:5000), GAPDH (GTX627408 peroxidase coupled, Genetex, 1:20,000), SLC2A1 (MA5-31960, Invitrogen, 1:5000), SLC2A3 (PA5-72331, Thermo Fisher Scientific, 1:5000) were incubated in blocking buffer overnight at 4 °C.

Techniques: Functional Assay, Membrane, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Clone Assay, Derivative Assay

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