Journal:

Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures

doi:

Figure Lengend Snippet: vv early mRNAs and intracellular cores are aligned on microtubules by immunofluorescence. (A) Cells were infected and transfected with BrUTP for 2 h. Cells were extracted with TX-100 before fixation and double-labeling with either anti-tubulin and anti-BrU (left panel) to reveal the viral mRNA structures or anti-p39 (right panel) to label intracellular cores. The high-magnification view (indicated with 1 and 2) of the indicated areas 1 and 2 in the merge of anti-tubulin and p39 shows that intracellular cores are aligned on MT tracks (arrowheads). In B in the panel indicated with + nocodazole, cells were treated with 20 μM nocodazole 1 h before infection and BrUTP-transfection and fixed at 2 h postinfection in the continued presence of the drug. In the + latrunculin panel, cells were infected and transfected and treated 20 min before fixation with 1 μM Lat A and 2.5 μM taxol. Fixed cells were then double-labeled with anti-BrU and with either anti-tubulin or with rhodamine-phalloidin. Cells were fixed without prior TX-100 extraction. MTs (α-tubulin) as well as the viral mRNAs (α-BrU) show a diffuse pattern in the cytosol after nocodazole treatment. In the presence of Lat A, the phalloidin labeling is diffuse too, but the BrU pattern seems unaffected. Scale bars, 10 μm. In the table at the bottom of B, cells were infected and BrUTP-transfected for 2 h 20 min before fixation cells were mock treated or treated with either 40 μM of nocodazole or with 1 μM Lat A and 2.5 μM taxol. The cells were then extracted with TX-100, fixed, and labeled with anti-BrU. The amount of BrU-positive structure was counted in 30 infected/transfected cells. Because nocodazole-treated cells mostly lacked any BrU-positive structures, making it difficult to estimate which cells were transfected, cells that showed some residual BrU labeling were considered for counting. The values represent the average amount of BrU structures per cell and the SEM

Article Snippet: The membranes were blocked in PBS, 0.2% Tween 20, and 5% milk powder for 2 h before incubation with anti–β-tubulin or anti–β-actin antibodies followed by horseradish peroxidase-tagged goat anti-mouse antibody ( Bio-Rad ).

Techniques: Immunofluorescence, Infection, Transfection, Labeling

Journal:

Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures

doi:

Figure Lengend Snippet: Biochemical evidence for MT association of viral mRNAs. (A–C) Cells were infected for 2 h. They were either left untreated or were treated with either 20 μM of nocodazole from 1 h before infection onward or with 1 μM Lat A together with 2.5 μM taxol 20 min before cell lysis. (A and B) Total RNA was extracted from infected cells from either the detergent-soluble (S) or -insoluble cytoskeletal fraction (CK), and the fractions were analyzed by an RNase protection assay with the use of probes to H5R and β-actin mRNA. The position of the H5R (A) and β-actin (B) protected mRNA is indicated on the right. The percentage of protected mRNAs in the cytoskeleton (CK) versus soluble (S) fraction of eachsample as assessed by phosphoimager analysis is indicated at the bottom of each panel. (C) Western blot analysis of actin and tubulin (the position of the respective protein is indicated) after nocodazole or Lat A treatment in the soluble (S) and cytoskeleton (CK) fraction. The proteins were detected with the use of antibodies to β-actin or β-tubulin followed by enhanced chemiluminescence.

Article Snippet: The membranes were blocked in PBS, 0.2% Tween 20, and 5% milk powder for 2 h before incubation with anti–β-tubulin or anti–β-actin antibodies followed by horseradish peroxidase-tagged goat anti-mouse antibody ( Bio-Rad ).

Techniques: Infection, Lysis, Rnase Protection Assay, Western Blot

Journal:

Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures

doi:

Figure Lengend Snippet: P25 (4RL) colocalizes with the viral mRNAs early in infection and may associate with MTs. Top panels in A: Infected and BrUTP-transfected HeLa cell were fixed at the indicated times postinfection and double-labeled with anti-BrU and anti-p25 antibodies. At 30 min postinfection the viral mRNAs partially colocalize with the diffuse p25 labeling, whereas at 45 min postinfection colocalization of the two markers is detected in the perinuclear region where the mRNAs appear still unorganized. At 60 min postinfection, when all of the mRNAs are organized into the typical punctate structures, no colocalization is detected. In the merge anti-BrU is shown in green and anti-p25 in red. Note that over time the p25 labeling decreases. Bottom panels in B: Cells were extracted with TX-100 before fixation at 30 min postinfection and double-labeling with antitubulin and anti-p25. Anti-p25 labeling appears to follow MT tracks (arrowheads). In the merge p25 is in the green and antitubulin in the red channel. Scale bars, top, 10 μm; bottom, 2 μm. (B) Infected HeLa cells were mock treated or treated with either nocodazole or Lat A (together with taxol) and Triton-extracted before fixation at 1 h postinfection. The cells were then labeled with antibodies to β-tubulin, p25, or rhodamine-phalloidin. Ten random pictures of both nocodazole- and mock-treated cells were taken with the use of the same parameters (the same amplitude, gain, and offset). The total fluorescence for p25 or actin was measured with the use of NIH image. The graph represents the average and SD of the total fluorescence of 10 cells for both treated and untreated cells.

Article Snippet: The membranes were blocked in PBS, 0.2% Tween 20, and 5% milk powder for 2 h before incubation with anti–β-tubulin or anti–β-actin antibodies followed by horseradish peroxidase-tagged goat anti-mouse antibody ( Bio-Rad ).

Techniques: Infection, Transfection, Labeling, Fluorescence

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: Cells

Article Title: BNIP3/BNIP3L-Dependent Mitophagy Protects Against Hippocampal Neuronal Damage and Apoptosis in a Model of Vascular Dementia

doi: 10.3390/cells15070585

Figure Lengend Snippet: BNIP3 knockdown exacerbates OGD-induced neuronal injury by impairing mitophagy and mitochondrial function. ( A ) Representative blots showing p62, LC3-I/II, Beclin1, Tomm20, BNIP3, and BNIP3L expression. ( B ) Quantification of protein levels normalized to β-actin, GAPDH, or Tubulin. (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; Data = mean ± SD; n = 3 repetitions/group). ( C ) Representative WB of BNIP3 after siRNA transfection. ( D ) Quantification of BNIP3 protein levels (** p < 0.01 vs. siNC; Data = mean ± SD; n = 3 repetitions/group). ( E ) Cell viability (CCK-8 assay) in HT22 neurons under OGD with/without siBNIP3 (*** p < 0.001; Data = mean ± SD; n = 3 repetitions/group). ( F ) Representative TEM images of each group in HT22 cells (scale bar: 500 nm). ( G ) The ratio of autophagic structures to mitochondria across multiple fields from each group (* p < 0.05, ** p < 0.01, n = 3 repetitions /group). ( H ) Representative flow cytometry images of each group. ( I ) Quantification of ROS intensity (*** p < 0.001; Data = mean ± SD; n = 3 repetitions/group). ( J ) JC-1 aggregates (red, healthy) and monomers (green, depolarized). ( K ) Quantification of JC-1 green/red ratio (*** p < 0.001; Data = mean ± SD; n = 3 repetitions/group).

Article Snippet: The following primary antibodies were used: BNIP3 (Abways, CY6771, Shanghai, China), BNIP3L (Abways, CY6906), β-actin (Affinity, Cincinnati, OH, USA, AF7018), Tomm20 (Abways, CY5527), Beclin1 (Abways, CY5092), P62 (Abways, CY5546), LC3B (Abways, CY5992), PINK1 (Proteintech, 23274-1-AP, Wuhan, China), Parkin (Abways, CY6641), FUNDC1 (Biodragon, BD-PT5658, Suzhou, China), Bcl2 (Abways, CY6717), BAX (Abways, CY5059), Caspase3 (Abways, CY5501), GAPDH (Abways, AB0037), Tubulin (Boster, A01857-1, Wuhan, China).

Techniques: Knockdown, Expressing, Control, Transfection, CCK-8 Assay, Flow Cytometry

Journal: Molecular neurobiology

Article Title: Microtubule-Actin Crosslinking Factor 1 is required for dendritic arborization and axon outgrowth in the developing brain

doi: 10.1007/s12035-015-9508-4

Figure Lengend Snippet: (A) MACF1 expression in cortical neurons. Cortical neurons from E14.5 control (MACF1loxP/+; Nex-cre) and MACF1loxP/loxP; Nex-cre brains were cultured for 5 days, and MACF1 localization was examined using immunostaining. Arrows indicate an accumulation of MACF1 in dendritic tips (B) MACF1 deletion causes aberrant actin arrangement and localization. Polymerized actins were visualized by phalloidin staining. Top panels show the patterns of polymerized actins at neurite tips. Middel and bottom panels show polymerized actins accumulated in the cytosol. (C) The levels of polymerized actin (F-actin) intensity in the cell body and at the neurite tip were quantified using Image J (NIH) software. n = 21 cells from 3 independent cultures using 3 mice for each condition. Statistical significance was determined by two-tailed Student's t-test. ***p < 0.001. (D) MACF1-deleted neurons show abnormal microtubule arrangement at the neurite tip. (E) Immunoblotting was performed to measure the levels of α-tubulin or acetylated-tubulin using E14.5 control and MACF1loxP/loxP; Nex-cre brain lysates (top panel). The levels of each tubulin were quantified in the bottom panel. n = 3 blots using 3 different lysates from 3 mice for each condition. Statistical significance was determined by two-tailed Student's t-test. ***p < 0.001.

Article Snippet: The following primary antibodies were used: Chicken anti-GFP (Invitrogen), Rabbit anti-GFP (Invitrogen), mouse anti-β-III Tubulin (Phosphosolutions), rabbit anti-acetyl-α-tubulin (Cell Signaling), and mouse anti-β-tubulin (Upstate).

Techniques: Expressing, Control, Cell Culture, Immunostaining, Staining, Software, Two Tailed Test, Western Blot

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: Ventral midbrain NSPC isolated from E12 rat embryos are multipotent. (A) VM NSPC cultured in the presence of the mitogenic factor FGF-2 express the markers of undifferentiated cells: Sox2, Vimentin and Nestin in proliferation stage. (B) After 6 days without FGF-2 (differentiation stage), VM NSPC differentiate into the three lineages of Central Nervous System: neurons (β-III Tubulin + and MAP2+), astrocytes (GFAP+) and oligodendrocytes (O4+), confirming the multipotency of these cultures. Nuclei were stained with Hoechst. Scale bars = 100 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Isolation, Cell Culture, Staining

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA promotes neuronal differentiation but decreases dopaminergic neurons in cultured VM NSPC. After proliferation, cells were kept on differentiating conditions for 6 days (N2 medium without FGF-2) and treated daily with different concentrations of HA (from 1 μM to 1 mM HA). VM NSPCs were analyzed after differentiation. (A) Micrographs showing the neuronal marker β-III Tubulin in control and 10 μM HA-treated cultures. Nuclei were stained with Hoechst. (B) Quantification of β-III Tubulin labeled cells relative to the total number of cells, showing a significant increase in the proportion of β-III Tubulin + cells caused by treatment with 10 μM HA. (C) Micrographs of double immunocytochemistry to detect the neuronal marker β-tubulin III and the dopaminergic marker Tyrosine Hydroxylase (TH) in control and 1 mM HA-treated cultures. Nuclei were stained with Hoechst. Scale bar = 100 μm. (D) Graph showing the percentage of TH-positive neurons in control and after HA treatments, relative to the total number of β-Tubulin III-positive cells. TH + neurons were significantly decreased after treatment with 1 mM HA. **p < 0.01. (E) Micrographs showing the dopaminergic marker TH in control cells, and the decrease caused by 1 mM HA. The H1R antagonist chlorpheniramine was tested either with or without HA. Nuclei were stained with Hoechst. (F) HA-induced decrease of TH-positive numbers was antagonized by chlorpheniramine. Note that the percentage of TH-positive cells is lower than in Figure D because it is normalized by the total number of cells. *p < 0.05 relative to control; ## p < 0.01 compared to 1 mM HA. Scale bars = 100 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Cell Culture, Marker, Control, Staining, Labeling, Immunocytochemistry

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA injection decreases dopaminergic neurons without affecting β-III Tubulin. (A) Coronal sections of the ventral midbrain from E14 vehicle- and HA-injected rat embryos stained to detect neurons (β-III Tubulin+) and dopaminergic phenotype (TH+); a representative section of a vehicle-injected embryo showing many TH-positive neurons while in a similar midbrain section from a representative HA-injected embryo less TH-positive cells are present. Nuclei were detected with Hoechst. Inner white squares in the merge images represent the higher magnifications shown at the bottom. Scale bars: 150 μm and 50 μm for the low-power and high-power pictures, respectively. (B) β-III Tubulin and TH expression by qRT-PCR from E14 vehicle- and HA-injected embryos, with β-III Tubulin presenting no change, and a significant decrease in TH caused by HA. **p < 0.01. (C) Densitometric analyses of the effect of HA administration on protein levels of β-III Tubulin and diverse factors involved in dopaminergic specification and phenotype. HA injection decreased the protein level of TH, Lmx1a, Lmx1b and Pitx3 in midbrain tissue from E14 HA-injected embryos without affecting the generation of neurons, compared to the vehicle-injected condition. Values were normalized to GAPDH signal. *p < 0.05.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Expressing, Quantitative RT-PCR

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA does not alter GABAergic or serotoninergic neurons. (A) Coronal sections of the ventral midbrain from E14 vehicle- and HA-injected rat embryos stained to identify neurons (β-III Tubulin+) and GABA-synthesizing cells (anti-GAD65/67+ antibody); vehicle-injected embryos had the same proportion of GAD65/67+ cells when compared to HA-injected embryos. Nuclei were stained with Hoechst. Scale bar: 150 μm. (B) HA did not modify the level of GAD65/67 protein in E14 midbrain tissue compared to vehicle-injected embryos by Western blot. The graph represents the densitometric analysis of GAD65/67 protein content where no significant effects were found; values were normalized to GAPDH. (C) Coronal sections of the ventral midbrain from vehicle- and HA-injected E14 embryos stained with β-III Tubulin and serotonin antibodies, where no differences in serotoninergic neurons are observed. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Western Blot

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA acts on early dopamine neural precursors but differentiated dopamine neurons are resistant. Sagittal sections of the VM from vehicle- and HA-treated rat embryos that were injected at different developmental stages, and analyzed two days later. Sections were stained to detect neurons (β-III Tubulin+) and dopaminergic phenotype (TH+); nuclei were detected with Hoechst. The decreasing effect of HA injection on dopaminergic phenotype is evident only when injected at early developmental stages (E10-E12 or E12-E14) but not at later developmental stages (E14-E16 or E16-E18). For the vehicle-injected E10-E12 embryos, only a few TH + neurons were found in the isthmic region and these neurons were absent in HA-injected organisms. Scale bars are indicated in the figure.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: High doses of chlorpheniramine affect normal embryonic development but neither chlorpheniramine nor cimetidine affects dopaminergic neurons. (A) Micrographs from E14 rat embryos injected at E12 with different doses of the H 1 R antagonist, chlorpheniramine. Fifty and twenty-five micrograms interfered with normal development, while 15 μg allows normal development of the embryo. (B) TH and β-III Tubulin staining in coronal sections of the VM from E14 rat embryos injected with 15 μg of the H 1 R antagonist, chlorpheniramine or 50 μg of the H 2 R antagonist, cimetidine. The staining patterns were not modified by these antagonists. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Modification

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: The H 1 R antagonist chlorpheniramine, but not the H 2 R antagonist cimetidine, abolished the deleterious effect of HA administration on dopaminergic differentiation. (A-D) TH and β-III Tubulin staining in coronal sections of the VM from E14 rat embryos with the indicated treatments. (A) Normal pattern of dopaminergic neuron staining in a vehicle-injected embryo. (B) Decrease in TH immunoreactivity due to HA administration. (C) Protective effect of the H 1 R antagonist chlorpheniramine on HA-induced decrease of TH immunoreactivity. (D) Administration of HA and the H 2 R antagonist cimetidine did not modify dopaminergic neurons compared to HA-injected embryos. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Staining, Injection

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: Primer sequences for detection of transcripts by qRT-PCR

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: