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  • 93
    Santa Cruz Biotechnology glyceraldehyde 3 phosphate dehydrogenase gapdh
    Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors. (A) Quantitative RT-PCR of Wnt/ β -catenin target genes . Expression of Axin2 mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. Axin2 mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) Western blot for β-catenin protein . Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.
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    Santa Cruz Biotechnology anti bax
    <t>BAX</t> and <t>BAK</t> can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P
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    Santa Cruz Biotechnology glyceraldehyde 3 phosphate dehydrogenase
    Analysis of inflammation in control and dystrophic muscle cells. Immunoblot analysis of TNF-α (A) and NF-κB (B). Graphs show protein level in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). <t>Glyceraldehyde-3-phosphate</t> dehydrogenase (GAPDH) was used as a loading control. All the experiments were performed in triplicate, and the relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P
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    Santa Cruz Biotechnology rabbit anti bax
    Downregulated Mctp1 and <t>Rarb</t> are associated with apoptotic cell death. a <t>Bax</t> proteins were increased by double knockdown of Mctp1 and Rarb. Bcl2 expression was decreased by siMctp1 and Rarb. b Mctp1 transcripts were downregulated. c Knockdown of Mctp1 enhanced Intracellular Ca 2+ levels (siCont (0.063) versus siMctp1 (0.131) in fluorescence intensities from baseline 490/525 ratio). d Rarb transcripts were downregulated. e-f Transfected siRarb reduced neuronal cell differentiation (MAP2). Scale bar, 40 μm. g and h Double knockdown of Mctp1 and Rarb increased Bax transcripts and decreased Bcl2 transcripts. i Transfected siMctp1 and siRarb activated LDH release. j Flow cytometry verified that downregulated Mctp1 and Rarb significantly induce apoptotic cell death in mouse NSCs. Scrambled siRNA served as a negative control (siCont). Significantly different at *, p
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    Santa Cruz Biotechnology rabbit polyclonal anti bax
    Ku70 mediates <t>Bax</t> deubiquitylation. ( A ) Time-course analysis of ubiquitylated Bax levels by Western blot with <t>polyclonal</t> antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in Ku70 −/− cells. ( B ) In vitro deubiquitylation of Bax in Ku70 −/− cell extracts supplemented with PBS or recombinant Ku70-rKU70. ( C ) In vitro deubiquitylation of Bax in Ku70 −/− cells that were grown in the presence of the proteasomal inhibitor MG-132. The extracts were supplemented with PBS or rKu70. ( D ) Levels of ubiquitylated Bax in wild-type cells that were grown in medium supplemented with DMSO or the proteasomal inhibitor, MG-132. ( E ) In vitro deubiquitylation assay of homogenous tetraubiquitin chains supplemented with BSA and rKu70 for various time periods, demonstrating that Ku70 possess an intrinsic DUB enzymatic activity. Incubation of tetraubiquitin only or tetraubiquitin together with BSA did not induce hydrolysis of the tetraubiquitin chains into monoubiquitin units. In A–D , β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.
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    Santa Cruz Biotechnology gapdh
    Inhibition of HDAC6 blocks activation of <t>TGF-β1</t> signaling in the peritoneum induced by high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates was used for measuring TGF-β1 by the ELISA. (B) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against TGFβ-RI, p-Smad3, Smad3, or <t>GAPDH.</t> (C) Expression level of TGFβ-RI was quantified by densitometry and normalized with GAPDH. (D) Expression level of p-Smad3 was quantified by densitometry and normalized with total Smad3. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P
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    93
    Santa Cruz Biotechnology anti glyceraldehyde 3 phosphate dehydrogenase
    Human papillomavirus (HPV) E7 oncoproteins increase catalase protein levels and activity and decrease SOD1 protein levels and SOD activity. Representative immunoblot (A) and densitometric analysis (B) of catalase levels in C33A cells 48 hours after transfection with HA-tagged HPV16 E7, HIS-tagged HPV18 E7 or control pcDNA3 plasmids. Catalase enzymatic activity in cells harbouring HPV E7 oncoproteins (C). Representative immunoblot of SOD1 and SOD2 (D, F) and respective densitometric analysis (E, G) in C33A transfected cells. <t>Glyceraldehyde</t> 3 phosphate dehydrogenase (GAPDH) was used as a loading control. SOD enzymatic activity in C33A transfected cells is shown (H). Data is expressed as the mean±SD, Tukey's test *p
    Anti Glyceraldehyde 3 Phosphate Dehydrogenase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti gapdh
    Inhibition of SIRT1 expression enhances VSVΔM51 infectivity and cell death in PC-3 cells. (A and B) PC-3 cells were treated with SAHA (5 μM), RESM (5 μM), TBSA (10 μM), or MS-275 (10 μM) for 24 h or were left untreated (DMSO) and were subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ) for 24 h. (A) Total cell extracts were analyzed by immunoblotting for SIRT1, IκB α, and <t>VSV</t> G. β-Actin was used as a loading control. Results are from a representative experiment. (B) Analysis of Sirt1 gene expression by qPCR. Gene expression level was calculated using the ΔΔ C T method. Data represent means ± SD of results from three independent experiments. (C and D) PC-3 cells were treated with increasing concentrations of nicotinamide (NAM) for 24 h and subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ) for 24 h. (C) Infectivity was determined at 24 h p.i. by fluorescence microscopy or quantified by flow cytometry. (D) Total cell extracts were analyzed by immunoblotting for SIRT1, p21, p62/SQSTM1, and LC3B. <t>GAPDH</t> was used as a loading control. Results are from a representative experiment. (E to I) PC-3 cells were transfected with control (CTR) or Sirt1 siRNA and were infected 48 h later with VSVΔM51-GFP (MOI of 0.01 or 0.05). (E) Whole-cell extracts were analyzed by immunoblotting for SIRT1 and VSV G. GAPDH was used as a loading control. Results are from a representative experiment. Lipo, Lipofectamine; siCTR, small interfering control; siSIRT1, small interfering SIRT1. (F and G) Viral infectivity was determined at 24 h p.i. by fluorescence microscopy (F) and measured by flow cytometry (G). Data represent means ± SD of results from three independent experiments. (H) Viral RNA levels were measured by qPCR. (I) Cell death was assessed using 7-AAD staining by flow cytometry.
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    Santa Cruz Biotechnology anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody
    UA sensitizes HepG2/DDP cells to low-dose cisplatin via inhibition of Nrf2/ARE signaling pathway. Notes: ( A ) HepG2/DDP cells were transfected with Nrf2 siRNA (si-Nrf2) or negative control (si-Con), or ( C ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with 8.92 μg/mL cisplatin (IC 30 of cisplatin for HepG2/DDP cells) and/or UA (2.25 μg/mL) for 48 hours. The level of Nrf2, HO-1, NQO1, and GST was detected by Western blot analysis. ( B ) HepG2/DDP cells were transfected with si-Nrf2 or si-Con, or ( D ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with series concentration of cisplatin (2–512 μg/mL) and/or UA (2.25 μg/mL) for 48 hours. The inhibition rate of cell was detected by CCK8 assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: ARE, antioxidant response element; CCK8, Cell Counting Kit 8; cDNA, complementary DNA; <t>GAPDH,</t> <t>glyceraldehyde-3-phosphate</t> dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin–resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; IC 30 , 30% inhibitory concentration; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; siRNA, small interfering RNA; UA, ursolic acid.
    Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti bax
    Inhibition of ER stress-related apoptosis in the sciatic nerve of DPN rats by intrathecal injection of IRE1α siRNA. ( A ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against P-IRE1α and XBP-1s. β-actin was probed as loading control. The intensity is expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ( B ) TUNEL, p-JNK, and Caspae-12 were effectively eliminated as measured by immunohistochemistry method. Data are expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with Tamhane’s T2 analysis or unpaired Student’s t-test. ( C ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against GRP78, CHOP, Bcl-2, <t>Bax</t> and <t>Cleaved-Caspase-3.</t> β-actin was probed as loading control. Results of are expressed as mean ± SEM indicated as percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis (GRP78, Cleaved-Caspase-3, CHOP) or Tamhane’s T2 analysis (Bcl-2, Bax) or unpaired Student’s t-test. Δ P
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    Santa Cruz Biotechnology anti bax pab
    Interaction of <t>Bax</t> with Bcl-2 in BL2 EBV (−) and BL2/B95 EBV (+) cells untreated or treated with nutlin-3. Cells were treated with 10 μ M nutlin-3 or the solvent dimethyl sulfoxide for 7 h. Lysates were subjected to immunoprecipitation with agarose-conjugated anti-Bax <t>pAb</t> (IP) or agarose-conjugated control rabbit IgG (IgG control) and then subjected to western blotting with an anti-Bcl-2 mAb or an anti-Bax pAb. As a control for protein levels before IP, a portion of cell lysate (input) corresponding to 15% of the input for IP was also included in the western blot. All results are representative of three independent experiments
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    Santa Cruz Biotechnology mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh
    SPL treatment prevents diabetes-induced glomerular and tubular damage. (A) Schematic representation of the experimental strategy used in this study. Four groups of rats were studied: Control (CTL), Diabetic (DBT), DBT+SPL and SPL, respectively (for detail see Material and methods ). (B) Purity of isolated glomerular and proximal tubular segments was evaluated by IB analysis of Wilm´s tumor 1 (WT1) and dipeptidylpeptidase (DppD) expressions, respectively. (C) IB analysis of nephrin and WT1 shows that SPL treatment prevents diabetes-induced glomerular damage; densitometric analysis of nephrin (D) and WT1 (E) are shown ( S1 Fig ). (F) IB analysis of kidney injury molecule 1 (Kim)-1 and heat shock protein of 72 KDa (Hsp72) show that SPL treatment decreases diabetes-induced proximal tubular damage, densitometric analysis of Kim-1 (G) and Hsp72 (H) are shown ( S1 Fig ). No changes were found in SPL group compared to CTL group. (I) IF analysis confirms that SPL treatment decreases diabetes-induced Kim-1 expression (green label) in PT labeled with DppD (red label). Nuclei were marked with 4', 6-diamidino- 2-phenylindole (DAPI, blue label). No significant changes were found between SPL and CTL groups. Glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> was used as loading control for IB. Data are mean±SEM from 3 rats per group. *p
    Mouse Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology polyclonal anti bax
    <t>Bax</t> translocates to mitochondria and forms high mol wt oligomers during anoikis. (a) FSK-7 cells, adherent or detached (maintained on poly-HEMA for 15 min or 4 h), were separated into soluble (S) and membrane (M) fractions before SDS-PAGE and immunoblotting with <t>polyclonal</t> anti-Bax (top). 4 mM CHAPS was added, and Bax was immunoprecipitated with anti-Bax 62M. Immunoprecipitates were separated by SDS-PAGE and were immunoblotted with anti-Bax 5B7 (bottom). (b) Cells expressing the OMM marker YFP-XT were detached for various times before cytospinning, and were immunostained with the activation-dependent antibody 62M. 62M reactivity is seen at discrete regions on mitochondria at all time points. Bar, 5 μm. (c) Soluble and membrane fractions from adherent cells or cells maintained on poly-HEMA for 4 h were isolated as above. CHAPS was added to a final concentration of 4 mM, and the fractions were concentrated. 5 mg protein from each fraction was loaded onto a Sephacryl S100-HR column (see Materials and methods). Separated fractions were analyzed by SDS-PAGE and immunoblotting for Bax. (d) Membrane fractions from FSK-7 cells detached for 4 h were separated by size-exclusion chromatography as above. Fractions containing high (oligo) and low (mono) mol wt Bax complexes were collected. Fractions were concentrated and separated into three equal parts. These were treated with BS3 (with or without addition of 0.1% Triton X-100). Samples were separated by SDS-PAGE and were immunoblotted for Bax. Cytosolic Bax from adherent cells (Adh) was run for comparison after identical treatment. (e) Membrane fractions isolated from adherent cells or cells detached for various times and separated by size-exclusion chromatography were analyzed by SDS-PAGE and immunoblotting for Bax. Total soluble (S) and membrane (M) fractions show Bax translocation at each time point. (f) Membrane fractions prepared from adherent cells or cells detached for 15 min, 1 h, or 4 h were spilt into three and treated with 5 mM BS3 in the presence or absence of 0.1% Triton-X100. Samples were analyzed by SDS-PAGE and were immunoblotted for Bax.
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    Santa Cruz Biotechnology anti bax monoclonal antibody
    Photomicrographies showing the experimental group of rat tongue carcinogenesis after 4 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for <t>bax</t> (c) immunohistochemistry for <t>bcl-2;</t> (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)
    Anti Bax Monoclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology mouse anti glyceraldehyde 3 phosphate dehydrogenase
    Photomicrographies showing the experimental group of rat tongue carcinogenesis after 4 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for <t>bax</t> (c) immunohistochemistry for <t>bcl-2;</t> (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)
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    Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors. (A) Quantitative RT-PCR of Wnt/ β -catenin target genes . Expression of Axin2 mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. Axin2 mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) Western blot for β-catenin protein . Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.

    Journal: PLoS ONE

    Article Title: Wnt5a Suppresses Tumor Formation and Redirects Tumor Phenotype in MMTV-Wnt1 Tumors

    doi: 10.1371/journal.pone.0113247

    Figure Lengend Snippet: Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors. (A) Quantitative RT-PCR of Wnt/ β -catenin target genes . Expression of Axin2 mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. Axin2 mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) Western blot for β-catenin protein . Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.

    Article Snippet: To ensure equal protein loading between lanes, the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz) was determined for each blot.

    Techniques: Expressing, Quantitative RT-PCR, Software, Western Blot

    Wnt5a expressing tumors demonstrate a decrease in markers of the basal tumor subtype. (A) Western blot using protein lysates isolated from the epithelium of MMTV-Wnt1 and MMTV-1;MMTV-Wnt5a mammary tumors. The expression of molecular markers of basal and luminal tumor subtypes were compared. Keratin 6 and Keratin 5 were strongly down-regulated in Wnt5a expressing tumors. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B–D) Immunostaining for K6 . Sections from MMTV-Wnt1 (B) and MMTV-1;MMTV-Wnt5a (C) tumors were stained with anti-Keratin 6 antibody using immunofluorescence (K6 = green; nuclei = blue). The percentage of cells expressing K6 was determined and graphed (D). Values are means +/− standard error (n = 6 MMTV-Wnt1, 3 fields per tumor; n = 5 MMTV-Wnt1;MMTV-Wnt5a, 3 fields per tumor). MMTV-Wnt1;MMTV-Wnt5a tumors demonstrated a significant decrease in K6-expressing cells as measured by T-test (* = p

    Journal: PLoS ONE

    Article Title: Wnt5a Suppresses Tumor Formation and Redirects Tumor Phenotype in MMTV-Wnt1 Tumors

    doi: 10.1371/journal.pone.0113247

    Figure Lengend Snippet: Wnt5a expressing tumors demonstrate a decrease in markers of the basal tumor subtype. (A) Western blot using protein lysates isolated from the epithelium of MMTV-Wnt1 and MMTV-1;MMTV-Wnt5a mammary tumors. The expression of molecular markers of basal and luminal tumor subtypes were compared. Keratin 6 and Keratin 5 were strongly down-regulated in Wnt5a expressing tumors. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B–D) Immunostaining for K6 . Sections from MMTV-Wnt1 (B) and MMTV-1;MMTV-Wnt5a (C) tumors were stained with anti-Keratin 6 antibody using immunofluorescence (K6 = green; nuclei = blue). The percentage of cells expressing K6 was determined and graphed (D). Values are means +/− standard error (n = 6 MMTV-Wnt1, 3 fields per tumor; n = 5 MMTV-Wnt1;MMTV-Wnt5a, 3 fields per tumor). MMTV-Wnt1;MMTV-Wnt5a tumors demonstrated a significant decrease in K6-expressing cells as measured by T-test (* = p

    Article Snippet: To ensure equal protein loading between lanes, the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz) was determined for each blot.

    Techniques: Expressing, Western Blot, Isolation, Immunostaining, Staining, Immunofluorescence

    Ectopic expression of Wnt5a results in low expression of K6 and K14. Expression of molecular markers for basal and luminal progenitors in MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mammary glands was compared by western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Each lane contains protein isolated from a separate mouse.

    Journal: PLoS ONE

    Article Title: Wnt5a Suppresses Tumor Formation and Redirects Tumor Phenotype in MMTV-Wnt1 Tumors

    doi: 10.1371/journal.pone.0113247

    Figure Lengend Snippet: Ectopic expression of Wnt5a results in low expression of K6 and K14. Expression of molecular markers for basal and luminal progenitors in MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mammary glands was compared by western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Each lane contains protein isolated from a separate mouse.

    Article Snippet: To ensure equal protein loading between lanes, the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz) was determined for each blot.

    Techniques: Expressing, Western Blot, Isolation

    Wnt/β-catenin signaling is not down-regulated in Wnt5a expressing MMTV-Wnt1 glands. (A) Western blot analysis of β-catenin protein in MMTV-Wnt1mammary gland . The level of active β-catenin was compared in protein lysates from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mammary glands. Total β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin-to-β-catenin is shown at the bottom on the gel. Each lane represents a sample from a different mouse. (B) Quantitative RT-PCR of Axin2, a Wnt/β-catenin target gene, in unsorted primary mammary epithelial cells . Expression of Axin2 and hWnt5a m RNA in unsorted PMECs from MMTV-Wnt1;MMTV-Wnt5a vs. MMTV-Wnt1 mammary glands was determined by quantitative RT-PCR (n = 12 MMTV-Wnt1, n = 11 MMTV-Wnt1;MMTV-Wnt5a separate mice). Data are shown as tables obtained using REST analysis software. Expression = fold difference in MMTV-Wnt1;Wnt5a relative to MMTV-Wnt1 controls after normalization to Gapdh. (C) Quantitative RT-PCR of Axin2 in E-cadherin negative primary mammary epithelial cells . Expression of Axin2 and hWnt5a mRNA in E-cadherin (Ecad) negative PMECs from MMTV-Wnt1;MMTV-Wnt5a vs. MMTV-Wnt1 mammary glands was determined by quantitative RT-PCR (n = 2 MMTV-Wnt1, n = 2 MMTV-Wnt1;MMTV-Wnt5a separate mice). Data are shown as tables obtained using REST analysis software. Expression = fold difference in MMTV-Wnt1;Wnt5a relative to MMTV-Wnt1 controls after normalization to Gapdh.

    Journal: PLoS ONE

    Article Title: Wnt5a Suppresses Tumor Formation and Redirects Tumor Phenotype in MMTV-Wnt1 Tumors

    doi: 10.1371/journal.pone.0113247

    Figure Lengend Snippet: Wnt/β-catenin signaling is not down-regulated in Wnt5a expressing MMTV-Wnt1 glands. (A) Western blot analysis of β-catenin protein in MMTV-Wnt1mammary gland . The level of active β-catenin was compared in protein lysates from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mammary glands. Total β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin-to-β-catenin is shown at the bottom on the gel. Each lane represents a sample from a different mouse. (B) Quantitative RT-PCR of Axin2, a Wnt/β-catenin target gene, in unsorted primary mammary epithelial cells . Expression of Axin2 and hWnt5a m RNA in unsorted PMECs from MMTV-Wnt1;MMTV-Wnt5a vs. MMTV-Wnt1 mammary glands was determined by quantitative RT-PCR (n = 12 MMTV-Wnt1, n = 11 MMTV-Wnt1;MMTV-Wnt5a separate mice). Data are shown as tables obtained using REST analysis software. Expression = fold difference in MMTV-Wnt1;Wnt5a relative to MMTV-Wnt1 controls after normalization to Gapdh. (C) Quantitative RT-PCR of Axin2 in E-cadherin negative primary mammary epithelial cells . Expression of Axin2 and hWnt5a mRNA in E-cadherin (Ecad) negative PMECs from MMTV-Wnt1;MMTV-Wnt5a vs. MMTV-Wnt1 mammary glands was determined by quantitative RT-PCR (n = 2 MMTV-Wnt1, n = 2 MMTV-Wnt1;MMTV-Wnt5a separate mice). Data are shown as tables obtained using REST analysis software. Expression = fold difference in MMTV-Wnt1;Wnt5a relative to MMTV-Wnt1 controls after normalization to Gapdh.

    Article Snippet: To ensure equal protein loading between lanes, the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz) was determined for each blot.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Mouse Assay, Software

    BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: BAX and BAK can be autoactivated by DNA damage independently of activators BID, BIM, PUMA and NOXA through downregulation of BCL-2, BCL-X L and MCL-1 (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase, Bad, Bmf or Bik . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (b) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were infected with retrovirus expressing GFP or the indicated BH3-only proteins to induce spontaneous apoptosis. NOXA denotes human NOXA. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide, tunicamycin (TC) or thapsigargin (TG), were subjected to immunoblot analysis using the indicated antibodies. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 18h, and subjected to immunoblot analysis using the indicated antibodies. (e) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide and/or MG132 for 36h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (f) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bcl-2, Bcl-x L and/or Mcl-1 to induce spontaneous apoptosis. After 2 days, cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (g) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were untreated or treated with etoposide in the presence of the pancaspase inhibitor Q-VD-OPh to preserve cell integrity upon apoptosis induction. After 24 h, cells were permeabilized with digitonin and subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. (h) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs transfected with scrambled siRNA or siRNA against Bcl-x L and Mcl-1 were subjected to limited trypsin proteolysis. The BAK cleavage products were detected by an anti-BAK (G23) immunoblot. **, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Transformation Assay, Infection, Expressing, shRNA, Luciferase, Staining, Transfection

    Noxa deficiency further protects Bid −/− Bim −/− Puma −/− mouse embryonic fibroblasts or small intestine from apoptosis (a) The mRNA levels of Noxa in the indicated tissues or cells were assessed by qRT-PCR. Data are normalized against 18S rRNA (mean ± s.d., n = 3 independent experiments). (b) Primary MEFs generated from E13.5 wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO mouse embryos were untreated, or cultured in the absence of serum or glucose for 3 days, or in the presence of tunicamycin (TC) or thapsigargin (TG) for 2 days. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (c and d) Apoptosis in the small intestinal crypts of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), Bid −/− Bim −/− Puma −/− Noxa −/− QKO (n = 2), or conditional Bax and Bak DKO (n = 2) mice at 8 to 17 weeks of age at 4 h after 18 Gy whole body irradiation was assessed by TUNEL staining (brown, magnification 400×). 300 small intestinal crypts from each mouse were analyzed. Representative light microscopy images are shown in (c). Scale bars, 50 µm. The number of TUNEL positive cells in the crypts was quantified and summarized in (d) (mean ± s.d.). (e) CD4 + T cells purified from the spleens of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), or Bid −/− Bim −/− Puma −/− Noxa −/− QKO mice (n = 3) at 8 to 10 weeks of age were cultured in the absence of cytokine, in the presence of etoposide, in the presence of dexamethasone, or after exposure to 2.5 Gy γ-irradiation. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d.). **, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: Noxa deficiency further protects Bid −/− Bim −/− Puma −/− mouse embryonic fibroblasts or small intestine from apoptosis (a) The mRNA levels of Noxa in the indicated tissues or cells were assessed by qRT-PCR. Data are normalized against 18S rRNA (mean ± s.d., n = 3 independent experiments). (b) Primary MEFs generated from E13.5 wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO mouse embryos were untreated, or cultured in the absence of serum or glucose for 3 days, or in the presence of tunicamycin (TC) or thapsigargin (TG) for 2 days. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (c and d) Apoptosis in the small intestinal crypts of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), Bid −/− Bim −/− Puma −/− Noxa −/− QKO (n = 2), or conditional Bax and Bak DKO (n = 2) mice at 8 to 17 weeks of age at 4 h after 18 Gy whole body irradiation was assessed by TUNEL staining (brown, magnification 400×). 300 small intestinal crypts from each mouse were analyzed. Representative light microscopy images are shown in (c). Scale bars, 50 µm. The number of TUNEL positive cells in the crypts was quantified and summarized in (d) (mean ± s.d.). (e) CD4 + T cells purified from the spleens of wild-type (n = 3), Bid −/− Bim −/− Puma −/− TKO (n = 3), or Bid −/− Bim −/− Puma −/− Noxa −/− QKO mice (n = 3) at 8 to 10 weeks of age were cultured in the absence of cytokine, in the presence of etoposide, in the presence of dexamethasone, or after exposure to 2.5 Gy γ-irradiation. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d.). **, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Quantitative RT-PCR, Generated, Cell Culture, Staining, Mouse Assay, Irradiation, TUNEL Assay, Light Microscopy, Purification

    Quadruple deficiency of Bid, Bim, Puma and Noxa abrogates apoptosis in transformed mouse embryonic fibroblasts triggered by growth factor deprivation and ER stress but not genotoxic stress (a–g) E1A/Ras-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in the absence of serum (a), glucose (b) or glutamine (c), or in the presence of tunicamycin (d), thapsigargin (e) or etoposide (f), or irradiated with UV-C (g). Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). (h) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in in the presence of tunicamycin, thapsigargin or etoposide, or irradiated with UV-C. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). *, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: Quadruple deficiency of Bid, Bim, Puma and Noxa abrogates apoptosis in transformed mouse embryonic fibroblasts triggered by growth factor deprivation and ER stress but not genotoxic stress (a–g) E1A/Ras-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in the absence of serum (a), glucose (b) or glutamine (c), or in the presence of tunicamycin (d), thapsigargin (e) or etoposide (f), or irradiated with UV-C (g). Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). (h) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, Bid −/− Bim −/− Puma −/− Noxa −/− QKO, or Bax −/− Bak −/− DKO MEFs were untreated, or cultured in in the presence of tunicamycin, thapsigargin or etoposide, or irradiated with UV-C. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). *, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Transformation Assay, Cell Culture, Irradiation, Staining

    DNA damage activates BAX and BAK-dependent mitochondrial apoptosis in transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO mouse embryonic fibroblasts (a) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide for the indicated times, were subjected to subcellular fractionation. Cytosolic and mitochondrial fractions were analyzed by anti-cytochrome c, anti-LDH and anti-VDAC1 immunoblots. LDH and VDAC1 serve as cytosolic and mitochondrial controls, respectively. (b) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide for the indicated times, were analyzed for Caspase-3/7 activities (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide (etop) or tunicamycin (TC), were analyzed by anti-PARP, anti-cleaved Caspase-3, and anti-actin immunoblots. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase or Apaf-1 , or transfected with scrambled siRNA (siScr) or siRNA against Cytochrome c or Caspase-9 . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (e) Mitochondria isolated from SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs untreated or treated with etoposide for 15 h (WT) or 36 h (QKO) were subjected to BMH crosslinking. The BAX and BAK homo-oligomers were detected by anti-BAX and anti-BAK immunoblots, respectively. (f) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bax and/or Bak . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). *, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: DNA damage activates BAX and BAK-dependent mitochondrial apoptosis in transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO mouse embryonic fibroblasts (a) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide for the indicated times, were subjected to subcellular fractionation. Cytosolic and mitochondrial fractions were analyzed by anti-cytochrome c, anti-LDH and anti-VDAC1 immunoblots. LDH and VDAC1 serve as cytosolic and mitochondrial controls, respectively. (b) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide for the indicated times, were analyzed for Caspase-3/7 activities (mean ± s.d., n = 3 independent experiments). (c) SV40-transformed wild-type, Bid −/− Bim −/− Puma −/− TKO, or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs, untreated or treated with etoposide (etop) or tunicamycin (TC), were analyzed by anti-PARP, anti-cleaved Caspase-3, and anti-actin immunoblots. (d) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were infected with retrovirus expressing shRNA against luciferase or Apaf-1 , or transfected with scrambled siRNA (siScr) or siRNA against Cytochrome c or Caspase-9 . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (e) Mitochondria isolated from SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs untreated or treated with etoposide for 15 h (WT) or 36 h (QKO) were subjected to BMH crosslinking. The BAX and BAK homo-oligomers were detected by anti-BAX and anti-BAK immunoblots, respectively. (f) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs were transfected with scrambled siRNA (siScr) or siRNA against Bax and/or Bak . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). *, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Transformation Assay, Fractionation, Western Blot, Infection, Expressing, shRNA, Luciferase, Transfection, Staining, Isolation

    BCL-X L is superior to BCL-2 and MCL-1 in preventing DNA damage-induced apoptosis due to its dual inhibition of BAX and BAK as well as higher protein stability (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, HA-BCL-2, HA-BCL-X L or HA-MCL-1 were subjected to anti-HA immunoprecipitation in 0.2% NP-40 or 1% CHAPS lysis buffer. The input (5%) and immunoprecipitates were analyzed by anti-BAX, anti-BAK, and anti-HA immunoblots. (b) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, HA-BCL-2, HA-BCL-X L or HA-MCL-1 were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of HA-BCL-2, HA-BCL-X L or HA-MCL-1 was detected by an anti-HA immunoblot with or without etoposide treatment for 12 h. (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, BCL-2 or BCL-X L were transfected with scrambled siRNA (siScr) or siRNA against Bax or Bak . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (d) SV40-transformed wild-type MEFs stably expressing HA-tagged BCL-2 or BCL-X L were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of HA-tagged BCL-2 and BCL-X L was detected by an anti-HA immunoblot. (e) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, wild-type BCL-X L , BCL-X L mutant 1 (F131V/D133A) or BCL-X L mutant 8 (G138E/R139L/I140N) were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of BCL-X L was analyzed by an anti-BCL-X L immunoblot. *, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: BCL-X L is superior to BCL-2 and MCL-1 in preventing DNA damage-induced apoptosis due to its dual inhibition of BAX and BAK as well as higher protein stability (a) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, HA-BCL-2, HA-BCL-X L or HA-MCL-1 were subjected to anti-HA immunoprecipitation in 0.2% NP-40 or 1% CHAPS lysis buffer. The input (5%) and immunoprecipitates were analyzed by anti-BAX, anti-BAK, and anti-HA immunoblots. (b) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, HA-BCL-2, HA-BCL-X L or HA-MCL-1 were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of HA-BCL-2, HA-BCL-X L or HA-MCL-1 was detected by an anti-HA immunoblot with or without etoposide treatment for 12 h. (c) SV40-transformed Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, BCL-2 or BCL-X L were transfected with scrambled siRNA (siScr) or siRNA against Bax or Bak . After 48 h, cells were untreated or treated with etoposide for 36 h. Cell death was quantified by annexin-V staining (mean ± s.d., n = 3 independent experiments). (d) SV40-transformed wild-type MEFs stably expressing HA-tagged BCL-2 or BCL-X L were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of HA-tagged BCL-2 and BCL-X L was detected by an anti-HA immunoblot. (e) SV40-transformed wild-type or Bid −/− Bim −/− Puma −/− Noxa −/− QKO MEFs stably expressing GFP, wild-type BCL-X L , BCL-X L mutant 1 (F131V/D133A) or BCL-X L mutant 8 (G138E/R139L/I140N) were untreated or treated with etoposide. Cell death was quantified by annexin-V staining at the indicated times (mean ± s.d., n = 3 independent experiments). The expression of BCL-X L was analyzed by an anti-BCL-X L immunoblot. *, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Inhibition, Transformation Assay, Stable Transfection, Expressing, Immunoprecipitation, Lysis, Western Blot, Staining, Transfection, Mutagenesis

    NOXA directly activates BAX and BAK independently of BID, BIM and PUMA (a) Mitochondria isolated from wild-type or Bax −/− Bak −/− MEFs were incubated with the indicated IVTT proteins generated using reticulocyte lysates or wheat germ extract (WGE) at 30°C for 30 min, after which the release of cytochrome c was quantified by ELISA assays (mean ± s.d., n = 3 independent experiments). (b) Isolated wild-type mitochondria were incubated with the indicated amounts of IVTT mouse NOXA protein (WGE) and the release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (c) Isolated wild-type mitochondria were incubated with the indicated IVTT proteins (WGE) and the release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (d) Radiolabeled IVTT NOXA or tBID protein was incubated with GST, GST-BAXΔC or GST-BAKΔC protein immobilized on glutathione beads. The precipitates and input were analyzed by Nu-PAGE and autoradiography. (e) Isolated wild-type mitochondria were incubated with the indicated IVTT proteins for 30 min then treated with BMH crosslinker. The BAX and BAK homo-oligomers were detected by anti-BAX and anti-BAK immunoblots, respectively. (f) Mitochondria isolated from wild-type or Bid −/− Bim −/− Puma −/− MEFs were incubated with IVTT NOXA (WGE) at 30°C for the indicated times and release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (g) Bid −/− Bim −/− Puma −/− MEFs were infected with the indicated retrovirus. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). The expression of the indicated proteins was detected by an anti-HA immunoblot. (h and i) ANTS/DPX (fluorophore/quencher)-encapsulated liposomes were incubated with recombinant BAX protein plus the indicated IVTT proteins generated using WGE. The release of entrapped fluorophore monitored with time is shown in (h) (mean ± s.d., n = 3 independent experiments). The release of entrapped fluorophore at 60 min is shown in (i) (mean ± s.d., n = 3 independent experiments). (j and k) ANTS/DPX-encapsulated liposomes were incubated with recombinant BAX protein plus the indicated IVTT proteins (WGE). The release of entrapped fluorophore monitored with time is shown in (j) (mean ± s.d., n = 3 independent experiments). The release of entrapped fluorophore at 60 min is shown in (k) (mean ± s.d., n =3 independent experiments). **, P

    Journal: Nature cell biology

    Article Title: An Interconnected Hierarchical Model of Cell Death Regulation by the BCL-2 Family

    doi: 10.1038/ncb3236

    Figure Lengend Snippet: NOXA directly activates BAX and BAK independently of BID, BIM and PUMA (a) Mitochondria isolated from wild-type or Bax −/− Bak −/− MEFs were incubated with the indicated IVTT proteins generated using reticulocyte lysates or wheat germ extract (WGE) at 30°C for 30 min, after which the release of cytochrome c was quantified by ELISA assays (mean ± s.d., n = 3 independent experiments). (b) Isolated wild-type mitochondria were incubated with the indicated amounts of IVTT mouse NOXA protein (WGE) and the release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (c) Isolated wild-type mitochondria were incubated with the indicated IVTT proteins (WGE) and the release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (d) Radiolabeled IVTT NOXA or tBID protein was incubated with GST, GST-BAXΔC or GST-BAKΔC protein immobilized on glutathione beads. The precipitates and input were analyzed by Nu-PAGE and autoradiography. (e) Isolated wild-type mitochondria were incubated with the indicated IVTT proteins for 30 min then treated with BMH crosslinker. The BAX and BAK homo-oligomers were detected by anti-BAX and anti-BAK immunoblots, respectively. (f) Mitochondria isolated from wild-type or Bid −/− Bim −/− Puma −/− MEFs were incubated with IVTT NOXA (WGE) at 30°C for the indicated times and release of cytochrome c was quantified (mean ± s.d., n = 3 independent experiments). (g) Bid −/− Bim −/− Puma −/− MEFs were infected with the indicated retrovirus. Cell death was quantified by annexin-V staining at 30 h (mean ± s.d., n = 3 independent experiments). The expression of the indicated proteins was detected by an anti-HA immunoblot. (h and i) ANTS/DPX (fluorophore/quencher)-encapsulated liposomes were incubated with recombinant BAX protein plus the indicated IVTT proteins generated using WGE. The release of entrapped fluorophore monitored with time is shown in (h) (mean ± s.d., n = 3 independent experiments). The release of entrapped fluorophore at 60 min is shown in (i) (mean ± s.d., n = 3 independent experiments). (j and k) ANTS/DPX-encapsulated liposomes were incubated with recombinant BAX protein plus the indicated IVTT proteins (WGE). The release of entrapped fluorophore monitored with time is shown in (j) (mean ± s.d., n = 3 independent experiments). The release of entrapped fluorophore at 60 min is shown in (k) (mean ± s.d., n =3 independent experiments). **, P

    Article Snippet: Antibodies used for immunoblot analysis are listed as followed: anti-BAK (NT, Upstate), anti-BAK (G23, Santa Cruz Biotechnology), anti-BAX (N-20, Santa Cruz Biotechnology), anti-HA (12CA5), anti-BCL-XL (#2762, Cell Signaling Technology), anti-BCL-2 (3F11), anti-MCL-1 (#600-401-394, Rockland), anti-cytochrome c (#556433, Pharmingen), anti-LDH (#100-1173, Rockland), anti-VDAC1 (ab16814, Abcam), anti-cleaved Caspase-3 (#9661, Cell Signaling Technology), anti-PARP (#9542, Cell Signaling Technology), anti-BAD (C-20, Santa Cruz), anti-APAF-1 (AB16941, Upstate Biotechnology), anti-Caspase-9 (#9504, Cell Signaling Technology), and anti-actin (Chemicon).

    Techniques: Isolation, Incubation, Generated, Enzyme-linked Immunosorbent Assay, Polyacrylamide Gel Electrophoresis, Autoradiography, Western Blot, Infection, Staining, Expressing, Recombinant

    TRIM55 silencing inhibits I/R- and miR-378a-3p inhibitor-induced apoptosis of H9C2 cardiomyocytes. H9C2 cardiomyocytes were transfected with three TRIM55-siRNAs (siRNA-1, siRNA-2, siRNA-3) or scramble siRNA (siNC). ( A , B ) TRIM55 expression was measured. H9C2 cardiomyocytes following I/R injury were transfected with the TRIM55-siRNA and/or miR-378a-3p inhibitor. ( C , D ) Cell apoptosis was measured by flow cytometry. ( E – H ) Expression of TRIM55, DUSP1, JNK1/2, cleaved PARP and caspase-3, Bax, and Bcl-2 was measured. *** P

    Journal: Aging (Albany NY)

    Article Title: miR-378a-3p inhibits ischemia/reperfusion-induced apoptosis in H9C2 cardiomyocytes by targeting TRIM55 via the DUSP1-JNK1/2 signaling pathway

    doi: 10.18632/aging.103106

    Figure Lengend Snippet: TRIM55 silencing inhibits I/R- and miR-378a-3p inhibitor-induced apoptosis of H9C2 cardiomyocytes. H9C2 cardiomyocytes were transfected with three TRIM55-siRNAs (siRNA-1, siRNA-2, siRNA-3) or scramble siRNA (siNC). ( A , B ) TRIM55 expression was measured. H9C2 cardiomyocytes following I/R injury were transfected with the TRIM55-siRNA and/or miR-378a-3p inhibitor. ( C , D ) Cell apoptosis was measured by flow cytometry. ( E – H ) Expression of TRIM55, DUSP1, JNK1/2, cleaved PARP and caspase-3, Bax, and Bcl-2 was measured. *** P

    Article Snippet: The anti-Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Transfection, Expressing, Flow Cytometry

    DUSP1 overexpression inhibits I/R- and TRIM55 overexpression-induced apoptosis of H9C2 cardiomyocytes. H9C2 cardiomyocytes following I/R injury were transduced with a TRIM55 and/or DUSP1 expression vector or blank vector. ( A , B ) Cell apoptosis was measured by flow cytometry. ( C – H ) The expression of DUSP1, JNK1/2, JNK1/2, cleavage of PARP and caspase-3, Bax, and Bcl-2 was also measured. *** P

    Journal: Aging (Albany NY)

    Article Title: miR-378a-3p inhibits ischemia/reperfusion-induced apoptosis in H9C2 cardiomyocytes by targeting TRIM55 via the DUSP1-JNK1/2 signaling pathway

    doi: 10.18632/aging.103106

    Figure Lengend Snippet: DUSP1 overexpression inhibits I/R- and TRIM55 overexpression-induced apoptosis of H9C2 cardiomyocytes. H9C2 cardiomyocytes following I/R injury were transduced with a TRIM55 and/or DUSP1 expression vector or blank vector. ( A , B ) Cell apoptosis was measured by flow cytometry. ( C – H ) The expression of DUSP1, JNK1/2, JNK1/2, cleavage of PARP and caspase-3, Bax, and Bcl-2 was also measured. *** P

    Article Snippet: The anti-Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Over Expression, Transduction, Expressing, Plasmid Preparation, Flow Cytometry

    The miR-378a-3p mimic inhibits I/R-induced apoptosis in rats. Myocardial I/R model rats were injected with 50 mg/kg of the miR-378a-3p mimic or negative control (NC) 24 h before LCA ligation. ( A – C ) Histological assessment of the myocardium was performed by H E staining and TUNEL. Scale bar: 100 μm. ( D – F ) The expression of miR-378a-3p, TRIM55, Bax, and Bcl-2 was measured by real-time PCR. ( G ) The expression of TRIM55, DUSP1, p-JNK1/2, JNK1/2, cleavage of PARP and caspase-3, Bax, and Bcl-2 was measured by western blotting. *** P

    Journal: Aging (Albany NY)

    Article Title: miR-378a-3p inhibits ischemia/reperfusion-induced apoptosis in H9C2 cardiomyocytes by targeting TRIM55 via the DUSP1-JNK1/2 signaling pathway

    doi: 10.18632/aging.103106

    Figure Lengend Snippet: The miR-378a-3p mimic inhibits I/R-induced apoptosis in rats. Myocardial I/R model rats were injected with 50 mg/kg of the miR-378a-3p mimic or negative control (NC) 24 h before LCA ligation. ( A – C ) Histological assessment of the myocardium was performed by H E staining and TUNEL. Scale bar: 100 μm. ( D – F ) The expression of miR-378a-3p, TRIM55, Bax, and Bcl-2 was measured by real-time PCR. ( G ) The expression of TRIM55, DUSP1, p-JNK1/2, JNK1/2, cleavage of PARP and caspase-3, Bax, and Bcl-2 was measured by western blotting. *** P

    Article Snippet: The anti-Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Injection, Negative Control, Ligation, Staining, TUNEL Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    miR-378a-3p is upregulated in I/R-induced H9C2 cardiomyocytes and inhibits cell apoptosis. ( A ) miR-378a-3p expression was measured by Real-time PCR in H9C2 cardiomyocytes following 3 h ischemia and 6, 12 or 24 h reperfusion. H9C2 cardiomyocytes following 3 h ischemia and 24 h reperfusion were transfected with a miR-378a-3p mimic, inhibitor or negative control (NC) and those without I/R injury were used as a control. ( B ) miR-378a-3p expression, ( C , D ) cell apoptosis, and ( E – J ) protein expression of DUSP1, p-JNK1/2, JNK1/2, cleaved PARP and caspase-3, Bax, and Bcl-2 were measured. *** P

    Journal: Aging (Albany NY)

    Article Title: miR-378a-3p inhibits ischemia/reperfusion-induced apoptosis in H9C2 cardiomyocytes by targeting TRIM55 via the DUSP1-JNK1/2 signaling pathway

    doi: 10.18632/aging.103106

    Figure Lengend Snippet: miR-378a-3p is upregulated in I/R-induced H9C2 cardiomyocytes and inhibits cell apoptosis. ( A ) miR-378a-3p expression was measured by Real-time PCR in H9C2 cardiomyocytes following 3 h ischemia and 6, 12 or 24 h reperfusion. H9C2 cardiomyocytes following 3 h ischemia and 24 h reperfusion were transfected with a miR-378a-3p mimic, inhibitor or negative control (NC) and those without I/R injury were used as a control. ( B ) miR-378a-3p expression, ( C , D ) cell apoptosis, and ( E – J ) protein expression of DUSP1, p-JNK1/2, JNK1/2, cleaved PARP and caspase-3, Bax, and Bcl-2 were measured. *** P

    Article Snippet: The anti-Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control

    Analysis of inflammation in control and dystrophic muscle cells. Immunoblot analysis of TNF-α (A) and NF-κB (B). Graphs show protein level in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. All the experiments were performed in triplicate, and the relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P

    Journal: PLoS ONE

    Article Title: Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress

    doi: 10.1371/journal.pone.0128567

    Figure Lengend Snippet: Analysis of inflammation in control and dystrophic muscle cells. Immunoblot analysis of TNF-α (A) and NF-κB (B). Graphs show protein level in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. All the experiments were performed in triplicate, and the relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P

    Article Snippet: The following primary antibodies were used for Western blotting: (1) Dystrophin (mouse monoclal, Vector Laboratories, Ontario, Canadá); (2) NF-κB (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (3) TNF-α (rabbit anti-mouse polyclonal, Chemical, USA); (4) 4-HNE (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (5) MyoD (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (6) Anti-Skeletal Myosin (mouse monoclonal, Sigma; Saint Louis, USA); (7) glyceraldehyde-3-phosphate dehydrogenase (GAPDH; rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, California).

    Techniques:

    Analysis of oxidative stress in control and dystrophic muscle cells. In (A), immunoblot analysis shows several bands of 4-HNE-protein adducts, ranging from 17 to 170 kDa. Graphs show protein level in the muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In (B), quantification of H 2 O 2 production in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). In (C) analysis of glutathione levels in the muscle cells from normal (Ctrl) and dystrophic culture cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). All the experiments were performed in triplicate and data expressed as mean ± SD. The relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P

    Journal: PLoS ONE

    Article Title: Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress

    doi: 10.1371/journal.pone.0128567

    Figure Lengend Snippet: Analysis of oxidative stress in control and dystrophic muscle cells. In (A), immunoblot analysis shows several bands of 4-HNE-protein adducts, ranging from 17 to 170 kDa. Graphs show protein level in the muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In (B), quantification of H 2 O 2 production in the muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). In (C) analysis of glutathione levels in the muscle cells from normal (Ctrl) and dystrophic culture cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). All the experiments were performed in triplicate and data expressed as mean ± SD. The relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P

    Article Snippet: The following primary antibodies were used for Western blotting: (1) Dystrophin (mouse monoclal, Vector Laboratories, Ontario, Canadá); (2) NF-κB (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (3) TNF-α (rabbit anti-mouse polyclonal, Chemical, USA); (4) 4-HNE (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (5) MyoD (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (6) Anti-Skeletal Myosin (mouse monoclonal, Sigma; Saint Louis, USA); (7) glyceraldehyde-3-phosphate dehydrogenase (GAPDH; rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, California).

    Techniques:

    MyoD and Myosin Heavy Chain analysis in control and dystrophic muscle cells. Immunoblot analysis of MyoD (A) and Myosin Heavy Chain (B) and graphs showing protein level in the primary muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. All the experiments were performed in triplicate, and the relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P

    Journal: PLoS ONE

    Article Title: Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress

    doi: 10.1371/journal.pone.0128567

    Figure Lengend Snippet: MyoD and Myosin Heavy Chain analysis in control and dystrophic muscle cells. Immunoblot analysis of MyoD (A) and Myosin Heavy Chain (B) and graphs showing protein level in the primary muscle cells from normal (Ctrl) and dystrophic primary muscle cells untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. All the experiments were performed in triplicate, and the relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P

    Article Snippet: The following primary antibodies were used for Western blotting: (1) Dystrophin (mouse monoclal, Vector Laboratories, Ontario, Canadá); (2) NF-κB (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (3) TNF-α (rabbit anti-mouse polyclonal, Chemical, USA); (4) 4-HNE (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (5) MyoD (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (6) Anti-Skeletal Myosin (mouse monoclonal, Sigma; Saint Louis, USA); (7) glyceraldehyde-3-phosphate dehydrogenase (GAPDH; rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, California).

    Techniques:

    Morphology and cell proliferation in control and dystrophic muscle cells. In (A), morphology of normal (Ctrl) and dystrophic primary muscle cultures untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). Day 1 shows undifferentiated dystrophin-deficient ( mdx ) muscle cells; day 3 shows maturation process in mdx muscle cells and day 6 shows complete morphological maturation with organized sarcomeric structures. In (B), immunoblot analysis of dystrophin and graph showing protein level in the primary muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In (C), cell proliferation was assessed by measurement of MTT assay in the primary muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. In (D), diameter myotubes from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. All the experiments were performed in triplicate, and the relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P

    Journal: PLoS ONE

    Article Title: Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress

    doi: 10.1371/journal.pone.0128567

    Figure Lengend Snippet: Morphology and cell proliferation in control and dystrophic muscle cells. In (A), morphology of normal (Ctrl) and dystrophic primary muscle cultures untreated ( mdx untreated) and LLLT treatment analyzed after 24 hours ( mdx LA 24) and 48 hours ( mdx LA 48). Day 1 shows undifferentiated dystrophin-deficient ( mdx ) muscle cells; day 3 shows maturation process in mdx muscle cells and day 6 shows complete morphological maturation with organized sarcomeric structures. In (B), immunoblot analysis of dystrophin and graph showing protein level in the primary muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In (C), cell proliferation was assessed by measurement of MTT assay in the primary muscle cells from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. In (D), diameter myotubes from Ctrl, mdx untreated, mdx LA 24 and mdx LA 48. All the experiments were performed in triplicate, and the relative value of the band intensity was quantified and normalized by the corresponding Ctrl. a P

    Article Snippet: The following primary antibodies were used for Western blotting: (1) Dystrophin (mouse monoclal, Vector Laboratories, Ontario, Canadá); (2) NF-κB (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (3) TNF-α (rabbit anti-mouse polyclonal, Chemical, USA); (4) 4-HNE (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (5) MyoD (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, California); (6) Anti-Skeletal Myosin (mouse monoclonal, Sigma; Saint Louis, USA); (7) glyceraldehyde-3-phosphate dehydrogenase (GAPDH; rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, California).

    Techniques: MTT Assay

    Downregulated Mctp1 and Rarb are associated with apoptotic cell death. a Bax proteins were increased by double knockdown of Mctp1 and Rarb. Bcl2 expression was decreased by siMctp1 and Rarb. b Mctp1 transcripts were downregulated. c Knockdown of Mctp1 enhanced Intracellular Ca 2+ levels (siCont (0.063) versus siMctp1 (0.131) in fluorescence intensities from baseline 490/525 ratio). d Rarb transcripts were downregulated. e-f Transfected siRarb reduced neuronal cell differentiation (MAP2). Scale bar, 40 μm. g and h Double knockdown of Mctp1 and Rarb increased Bax transcripts and decreased Bcl2 transcripts. i Transfected siMctp1 and siRarb activated LDH release. j Flow cytometry verified that downregulated Mctp1 and Rarb significantly induce apoptotic cell death in mouse NSCs. Scrambled siRNA served as a negative control (siCont). Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: Downregulated Mctp1 and Rarb are associated with apoptotic cell death. a Bax proteins were increased by double knockdown of Mctp1 and Rarb. Bcl2 expression was decreased by siMctp1 and Rarb. b Mctp1 transcripts were downregulated. c Knockdown of Mctp1 enhanced Intracellular Ca 2+ levels (siCont (0.063) versus siMctp1 (0.131) in fluorescence intensities from baseline 490/525 ratio). d Rarb transcripts were downregulated. e-f Transfected siRarb reduced neuronal cell differentiation (MAP2). Scale bar, 40 μm. g and h Double knockdown of Mctp1 and Rarb increased Bax transcripts and decreased Bcl2 transcripts. i Transfected siMctp1 and siRarb activated LDH release. j Flow cytometry verified that downregulated Mctp1 and Rarb significantly induce apoptotic cell death in mouse NSCs. Scrambled siRNA served as a negative control (siCont). Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Expressing, Fluorescence, Transfection, Cell Differentiation, Flow Cytometry, Negative Control

    Downregulated miR-18b (miR-18b-5p) by SOD1 mutations contributes apoptotic cell death in SOD1(G93A) Tg mice and fALS patient spinal cord tissues. a Hif1α, Mef2c and Bax expression were increased in G93A mice. Mctp1, Rarb and Bcl2 proteins were decreased in G93A mice. b mRNAs of Hif1α, Mef2c and Bax was highly expressed in G93A mice. Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in G93A mice. c miR-18b (miR-18b-5p) was deeply reduced and miR-206 was dramatically induced in G93A mice ( n = 5). d The protein levels of Hif1α, Mef2c and Bax was upregulated in fALS (G86S) patient (Cervical and lumber). Mctp1, Rarb and Bcl2 proteins were decreased in fALS (G86S) patient. Normal spinal cord tissues (Cervical (control 1 and 2) served as a negative control (Cont). e The transcripts of Hif1α, Mef2c and Bax was highly upregulated in fALS (G86S) patient (Cervicals). Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in fALS (G86S) patient. f miR-18b (miR-18b-5p) expression was importantly decreased and miR-206 was highly expressed in fALS (G86S) patient (Cervial and Lumber). Normal spinal cord tissues (Cervicals (control 1 and 2) served as a negative control (Cont). hSOD1 immunoreactivity on western blots of the insoluble fraction of the G93A mice and fALS (G86S) patients tissues. Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: Downregulated miR-18b (miR-18b-5p) by SOD1 mutations contributes apoptotic cell death in SOD1(G93A) Tg mice and fALS patient spinal cord tissues. a Hif1α, Mef2c and Bax expression were increased in G93A mice. Mctp1, Rarb and Bcl2 proteins were decreased in G93A mice. b mRNAs of Hif1α, Mef2c and Bax was highly expressed in G93A mice. Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in G93A mice. c miR-18b (miR-18b-5p) was deeply reduced and miR-206 was dramatically induced in G93A mice ( n = 5). d The protein levels of Hif1α, Mef2c and Bax was upregulated in fALS (G86S) patient (Cervical and lumber). Mctp1, Rarb and Bcl2 proteins were decreased in fALS (G86S) patient. Normal spinal cord tissues (Cervical (control 1 and 2) served as a negative control (Cont). e The transcripts of Hif1α, Mef2c and Bax was highly upregulated in fALS (G86S) patient (Cervicals). Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in fALS (G86S) patient. f miR-18b (miR-18b-5p) expression was importantly decreased and miR-206 was highly expressed in fALS (G86S) patient (Cervial and Lumber). Normal spinal cord tissues (Cervicals (control 1 and 2) served as a negative control (Cont). hSOD1 immunoreactivity on western blots of the insoluble fraction of the G93A mice and fALS (G86S) patients tissues. Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Mouse Assay, Expressing, Negative Control, Western Blot

    SOD1 mutation (G93A) induces apoptosis. a Western blot analysis verified the protein levels of Hif1α, Mef2c, Mctp1, Rarb, Bax, and Bcl2 in three different NSC-34 cell lines. b Quantification analysis of western blot. The data represent the average ± SEM of 3 separate experiments. Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: SOD1 mutation (G93A) induces apoptosis. a Western blot analysis verified the protein levels of Hif1α, Mef2c, Mctp1, Rarb, Bax, and Bcl2 in three different NSC-34 cell lines. b Quantification analysis of western blot. The data represent the average ± SEM of 3 separate experiments. Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Mutagenesis, Western Blot

    miR-206 post-transcriptionally regulates Mctp1 and Rarb and elevates apoptotic cell death. a and b Luciferases assay with 3′ UTR of Mctp1 showed that Mctp1 is target of miR-206. Mctp1 transcripts was downregulated by increased miR-206. c Intracellular Ca 2+ levels (Cont (0.069) versus miR-206 (0.122) in fluorescence intensities from baseline 490/525 ratio) was enhanced by miR-206. d miR-206 controlled Mctp1 and Rarb protein expression. e Luciferase assay with 3′ UTR of Rarb also verified that Rarb is target of miR-206. Rarb mRNAs was decreased by miR-206. Neuronal cell differentiation (MAP2) was reduced by miR-206. Scale bar, 40 μm. f and g Both Bax protein and mRNAs were increased by miR-206. Bcl2 proteins and mRNAs were decreased by miR-206. h miR-206 was overexpressed in contNSC-34 cells. i ) LDH release assay showed that miR-206 enhances apoptosis. j Flow cytometry analysis explained that overexpressed miR-206 activates apoptotic cell death in mouse NSCs. Empty vector served as a negative control (Cont). Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: miR-206 post-transcriptionally regulates Mctp1 and Rarb and elevates apoptotic cell death. a and b Luciferases assay with 3′ UTR of Mctp1 showed that Mctp1 is target of miR-206. Mctp1 transcripts was downregulated by increased miR-206. c Intracellular Ca 2+ levels (Cont (0.069) versus miR-206 (0.122) in fluorescence intensities from baseline 490/525 ratio) was enhanced by miR-206. d miR-206 controlled Mctp1 and Rarb protein expression. e Luciferase assay with 3′ UTR of Rarb also verified that Rarb is target of miR-206. Rarb mRNAs was decreased by miR-206. Neuronal cell differentiation (MAP2) was reduced by miR-206. Scale bar, 40 μm. f and g Both Bax protein and mRNAs were increased by miR-206. Bcl2 proteins and mRNAs were decreased by miR-206. h miR-206 was overexpressed in contNSC-34 cells. i ) LDH release assay showed that miR-206 enhances apoptosis. j Flow cytometry analysis explained that overexpressed miR-206 activates apoptotic cell death in mouse NSCs. Empty vector served as a negative control (Cont). Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Fluorescence, Expressing, Luciferase, Cell Differentiation, Lactate Dehydrogenase Assay, Flow Cytometry, Plasmid Preparation, Negative Control

    miR-18b (miR-18b-5p) regulates Hif1α and reduces apoptosis in mtNSC-34 cells. a Overexpressed miR-18b (miR-18b-5p) decreased Hif1α and Mef2c proteins. Both Mctp1 and Rarb expression were increased by miR-18b (miR-18b-5p). Downregulated Bax and upregulated Bcl2 by miR-18b (miR-18b-5p) diminished apoptosis in mtNSC-34 cells. b RT-qPCR analysis showed low expression of Hif1α and Mef2c mRNAs. c Mctp1 and Rarb transcripts were highly expressed by miR-18b (miR-18b-5p). d Bax mRNAs were decreased and Bcl2 mRNAs were increased by overexpressed miR-18b. e miR-18b (miR-18b-5p) was overexpressed in mtNSC-34 cells. f miR-206 was reduced by miR-18b (miR-18b-5p). g LDH release analysis explained that transfected miR-18b (miR-18b-5p) restores apoptosis. h Luciferases assay with 3′ UTR of Hif1α showed that Hif1α is target of miR-18b in contNSC-34 cells. i and j Overexpression of miR-18b (miR-18b-5p) enhanced neuronal differentiation (MAP2) and attenuated intracellular Ca 2+ levels (Cont (0.098) versus miR-18b (miR-18b-5p) (0.051) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Empty vector served as a negative control (Cont). Arrow represents SOD1 aggregation (green). Scale bar, 40 μm. Significantly different at *, p

    Journal: Translational Neurodegeneration

    Article Title: Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

    doi: 10.1186/s40035-020-00203-4

    Figure Lengend Snippet: miR-18b (miR-18b-5p) regulates Hif1α and reduces apoptosis in mtNSC-34 cells. a Overexpressed miR-18b (miR-18b-5p) decreased Hif1α and Mef2c proteins. Both Mctp1 and Rarb expression were increased by miR-18b (miR-18b-5p). Downregulated Bax and upregulated Bcl2 by miR-18b (miR-18b-5p) diminished apoptosis in mtNSC-34 cells. b RT-qPCR analysis showed low expression of Hif1α and Mef2c mRNAs. c Mctp1 and Rarb transcripts were highly expressed by miR-18b (miR-18b-5p). d Bax mRNAs were decreased and Bcl2 mRNAs were increased by overexpressed miR-18b. e miR-18b (miR-18b-5p) was overexpressed in mtNSC-34 cells. f miR-206 was reduced by miR-18b (miR-18b-5p). g LDH release analysis explained that transfected miR-18b (miR-18b-5p) restores apoptosis. h Luciferases assay with 3′ UTR of Hif1α showed that Hif1α is target of miR-18b in contNSC-34 cells. i and j Overexpression of miR-18b (miR-18b-5p) enhanced neuronal differentiation (MAP2) and attenuated intracellular Ca 2+ levels (Cont (0.098) versus miR-18b (miR-18b-5p) (0.051) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Empty vector served as a negative control (Cont). Arrow represents SOD1 aggregation (green). Scale bar, 40 μm. Significantly different at *, p

    Article Snippet: Samples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti-Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101-215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Over Expression, Fluorescence, Plasmid Preparation, Negative Control

    Ku70 mediates Bax deubiquitylation. ( A ) Time-course analysis of ubiquitylated Bax levels by Western blot with polyclonal antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in Ku70 −/− cells. ( B ) In vitro deubiquitylation of Bax in Ku70 −/− cell extracts supplemented with PBS or recombinant Ku70-rKU70. ( C ) In vitro deubiquitylation of Bax in Ku70 −/− cells that were grown in the presence of the proteasomal inhibitor MG-132. The extracts were supplemented with PBS or rKu70. ( D ) Levels of ubiquitylated Bax in wild-type cells that were grown in medium supplemented with DMSO or the proteasomal inhibitor, MG-132. ( E ) In vitro deubiquitylation assay of homogenous tetraubiquitin chains supplemented with BSA and rKu70 for various time periods, demonstrating that Ku70 possess an intrinsic DUB enzymatic activity. Incubation of tetraubiquitin only or tetraubiquitin together with BSA did not induce hydrolysis of the tetraubiquitin chains into monoubiquitin units. In A–D , β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation

    doi: 10.1073/pnas.0706700105

    Figure Lengend Snippet: Ku70 mediates Bax deubiquitylation. ( A ) Time-course analysis of ubiquitylated Bax levels by Western blot with polyclonal antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in Ku70 −/− cells. ( B ) In vitro deubiquitylation of Bax in Ku70 −/− cell extracts supplemented with PBS or recombinant Ku70-rKU70. ( C ) In vitro deubiquitylation of Bax in Ku70 −/− cells that were grown in the presence of the proteasomal inhibitor MG-132. The extracts were supplemented with PBS or rKu70. ( D ) Levels of ubiquitylated Bax in wild-type cells that were grown in medium supplemented with DMSO or the proteasomal inhibitor, MG-132. ( E ) In vitro deubiquitylation assay of homogenous tetraubiquitin chains supplemented with BSA and rKu70 for various time periods, demonstrating that Ku70 possess an intrinsic DUB enzymatic activity. Incubation of tetraubiquitin only or tetraubiquitin together with BSA did not induce hydrolysis of the tetraubiquitin chains into monoubiquitin units. In A–D , β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.

    Article Snippet: The following antibodies were used to detect protein expression by Western blot: rabbit polyclonal anti Bax (N-20; Santa Cruz Biotechnology), mouse monoclonal anti actin (A4700; Sigma), mouse monoclonal anti ubiquitin (P4D1; Santa Cruz Biotechnology), and mouse monoclonal anti HA (HA.11; Covance).

    Techniques: Western Blot, In Vitro, Recombinant, Activity Assay, Incubation, Modification

    Bax ubiquitylation promotes its degradation and decreases upon apoptotic stimuli. ( A ) Treatment of Ku70 −/− cells with the proteasomal inhibitor, MG-132, increased the levels of ubiquitylated Bax. ( B ) Treatment of HEK293 cells overexpressing siRNA against Ku70 with the proteasomal inhibitor Bertozomib (Velcade or PS-341) increased the levels of ubiquitylated Bax. The decrease in Ku70 levels was validated against HEK293 cells overexpressing control siRNA (data not shown) ( C ) Time-course analysis of ubiquitylated Bax levels by Western blot using polyclonal antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in wild-type cells. In all experiments, β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation

    doi: 10.1073/pnas.0706700105

    Figure Lengend Snippet: Bax ubiquitylation promotes its degradation and decreases upon apoptotic stimuli. ( A ) Treatment of Ku70 −/− cells with the proteasomal inhibitor, MG-132, increased the levels of ubiquitylated Bax. ( B ) Treatment of HEK293 cells overexpressing siRNA against Ku70 with the proteasomal inhibitor Bertozomib (Velcade or PS-341) increased the levels of ubiquitylated Bax. The decrease in Ku70 levels was validated against HEK293 cells overexpressing control siRNA (data not shown) ( C ) Time-course analysis of ubiquitylated Bax levels by Western blot using polyclonal antibody against Bax before (0) or at various times after the induction of Bax-mediated apoptosis by staurosporine-STS in wild-type cells. In all experiments, β-actin levels served as a loading control. U, unmodified Bax; M, modified Bax.

    Article Snippet: The following antibodies were used to detect protein expression by Western blot: rabbit polyclonal anti Bax (N-20; Santa Cruz Biotechnology), mouse monoclonal anti actin (A4700; Sigma), mouse monoclonal anti ubiquitin (P4D1; Santa Cruz Biotechnology), and mouse monoclonal anti HA (HA.11; Covance).

    Techniques: Western Blot, Modification

    Inhibition of HDAC6 blocks activation of TGF-β1 signaling in the peritoneum induced by high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates was used for measuring TGF-β1 by the ELISA. (B) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against TGFβ-RI, p-Smad3, Smad3, or GAPDH. (C) Expression level of TGFβ-RI was quantified by densitometry and normalized with GAPDH. (D) Expression level of p-Smad3 was quantified by densitometry and normalized with total Smad3. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Journal: Oncotarget

    Article Title: Histone deacetylase 6 inhibition counteracts the epithelial–mesenchymal transition of peritoneal mesothelial cells and prevents peritoneal fibrosis

    doi: 10.18632/oncotarget.20982

    Figure Lengend Snippet: Inhibition of HDAC6 blocks activation of TGF-β1 signaling in the peritoneum induced by high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates was used for measuring TGF-β1 by the ELISA. (B) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against TGFβ-RI, p-Smad3, Smad3, or GAPDH. (C) Expression level of TGFβ-RI was quantified by densitometry and normalized with GAPDH. (D) Expression level of p-Smad3 was quantified by densitometry and normalized with total Smad3. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Article Snippet: Antibodies to collagen I (A2), E-cadherin, TGF-βRI, CD68, EGFR, VEGF, CD31 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as well as HDAC6 siRNA were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

    Techniques: Inhibition, Activation Assay, Injection, Enzyme-linked Immunosorbent Assay, Expressing

    Inhibition of HDAC6 attenuates macrophage infiltration in the peritoneum induced by high glucose PDF Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against CD68 or GAPDH. (B) Photomicrographs (original magnification, ×200) illustrate CD68 staining of the peritoneal tissues. (C) The percentage of CD68-positive cells was calculated from ten random fields of six mice peritoneal samples. (D) Expression level of CD68 was quantified by densitometry and normalized with GAPDH. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Journal: Oncotarget

    Article Title: Histone deacetylase 6 inhibition counteracts the epithelial–mesenchymal transition of peritoneal mesothelial cells and prevents peritoneal fibrosis

    doi: 10.18632/oncotarget.20982

    Figure Lengend Snippet: Inhibition of HDAC6 attenuates macrophage infiltration in the peritoneum induced by high glucose PDF Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against CD68 or GAPDH. (B) Photomicrographs (original magnification, ×200) illustrate CD68 staining of the peritoneal tissues. (C) The percentage of CD68-positive cells was calculated from ten random fields of six mice peritoneal samples. (D) Expression level of CD68 was quantified by densitometry and normalized with GAPDH. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Article Snippet: Antibodies to collagen I (A2), E-cadherin, TGF-βRI, CD68, EGFR, VEGF, CD31 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as well as HDAC6 siRNA were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

    Techniques: Inhibition, Injection, Staining, Mouse Assay, Expressing

    Inhibition of HDAC6 reduces fibroblast activation and ECM protein deposition in the peritoneum after exposure to high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against α-SMA or GAPDH. (B) Expression levels of α-SMA were quantified by densitometry and normalized with GAPDH. (C) The percentage of collagen I-positive areas was calculated from ten random fields of six mice peritoneal samples. (D) Photomicrographs illustrate immunohistochemical staining of collagen I in the submesothelial compact zone (original magnification, ×200). Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Journal: Oncotarget

    Article Title: Histone deacetylase 6 inhibition counteracts the epithelial–mesenchymal transition of peritoneal mesothelial cells and prevents peritoneal fibrosis

    doi: 10.18632/oncotarget.20982

    Figure Lengend Snippet: Inhibition of HDAC6 reduces fibroblast activation and ECM protein deposition in the peritoneum after exposure to high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against α-SMA or GAPDH. (B) Expression levels of α-SMA were quantified by densitometry and normalized with GAPDH. (C) The percentage of collagen I-positive areas was calculated from ten random fields of six mice peritoneal samples. (D) Photomicrographs illustrate immunohistochemical staining of collagen I in the submesothelial compact zone (original magnification, ×200). Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Article Snippet: Antibodies to collagen I (A2), E-cadherin, TGF-βRI, CD68, EGFR, VEGF, CD31 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as well as HDAC6 siRNA were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

    Techniques: Inhibition, Activation Assay, Injection, Expressing, Mouse Assay, Immunohistochemistry, Staining

    Inhibition of HDAC6 abrogates the activation of EGFR/STAT3 signaling pathway in the peritoneum exposed to high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against p-EGFR, EGFR, p-STAT3, STAT3 or GAPDH. (B) Expression level of p-EGFR was quantified by densitometry and normalized with total EGFR. (D) Expression level of p-STAT3 was quantified by densitometry and normalized with total STAT3. Expression levels of total EGFR (C) or total STAT3 (E) were quantified by densitometry and normalized with total GAPDH. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Journal: Oncotarget

    Article Title: Histone deacetylase 6 inhibition counteracts the epithelial–mesenchymal transition of peritoneal mesothelial cells and prevents peritoneal fibrosis

    doi: 10.18632/oncotarget.20982

    Figure Lengend Snippet: Inhibition of HDAC6 abrogates the activation of EGFR/STAT3 signaling pathway in the peritoneum exposed to high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against p-EGFR, EGFR, p-STAT3, STAT3 or GAPDH. (B) Expression level of p-EGFR was quantified by densitometry and normalized with total EGFR. (D) Expression level of p-STAT3 was quantified by densitometry and normalized with total STAT3. Expression levels of total EGFR (C) or total STAT3 (E) were quantified by densitometry and normalized with total GAPDH. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Article Snippet: Antibodies to collagen I (A2), E-cadherin, TGF-βRI, CD68, EGFR, VEGF, CD31 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as well as HDAC6 siRNA were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

    Techniques: Inhibition, Activation Assay, Injection, Expressing

    Inhibition of HDAC6 reduces histone H3 acetylation in the peritoneum of mice after exposure to high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneum was taken for immunoblot analysis of acetyl histone H3, histone H3, acetyl α-tubulin, HDAC6 or GAPDH; the representative results with three samples are shown. (B) Expression level of acetyl-histone H3 was quantified by densitometry and normalized with total histone H3. (C and D) Expression levels of acetyl α-tubulin (C) or HDAC6 (D) were quantified by densitometry and normalized with GAPDH. (E) Photomicrographs illustrate co-staining of α-SMA and HDAC6 in the peritoneum collected 28 days after 4.25%PDF injection (original magnification, ×200). Data are represented as the means±SD (n=6). Means with different letters (a-c) are significantly different from one another (P

    Journal: Oncotarget

    Article Title: Histone deacetylase 6 inhibition counteracts the epithelial–mesenchymal transition of peritoneal mesothelial cells and prevents peritoneal fibrosis

    doi: 10.18632/oncotarget.20982

    Figure Lengend Snippet: Inhibition of HDAC6 reduces histone H3 acetylation in the peritoneum of mice after exposure to high glucose dialysate Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneum was taken for immunoblot analysis of acetyl histone H3, histone H3, acetyl α-tubulin, HDAC6 or GAPDH; the representative results with three samples are shown. (B) Expression level of acetyl-histone H3 was quantified by densitometry and normalized with total histone H3. (C and D) Expression levels of acetyl α-tubulin (C) or HDAC6 (D) were quantified by densitometry and normalized with GAPDH. (E) Photomicrographs illustrate co-staining of α-SMA and HDAC6 in the peritoneum collected 28 days after 4.25%PDF injection (original magnification, ×200). Data are represented as the means±SD (n=6). Means with different letters (a-c) are significantly different from one another (P

    Article Snippet: Antibodies to collagen I (A2), E-cadherin, TGF-βRI, CD68, EGFR, VEGF, CD31 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as well as HDAC6 siRNA were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

    Techniques: Inhibition, Mouse Assay, Injection, Expressing, Staining

    Inhibition of HDAC6 blocks activation of NF-κB signaling pathway and suppresses release of multiple inflammatory cytokines and chemokines Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against p-NF-κB, NF-κB or GAPDH. (B) Expression levels of p-NF-κB were quantified by densitometry and normalized with total NF-κB. Graphs show the expression levels of IL-6 (C) , TNF-α (D) , IL-1β (E) , and MCP-1 (F) by ELISA. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Journal: Oncotarget

    Article Title: Histone deacetylase 6 inhibition counteracts the epithelial–mesenchymal transition of peritoneal mesothelial cells and prevents peritoneal fibrosis

    doi: 10.18632/oncotarget.20982

    Figure Lengend Snippet: Inhibition of HDAC6 blocks activation of NF-κB signaling pathway and suppresses release of multiple inflammatory cytokines and chemokines Peritoneal membrane was collected at 28 days after PDF injection with or without administration of TA (70 mg/kg, daily). (A) The peritoneal tissue lysates were subjected to immunoblot analysis with specific antibodies against p-NF-κB, NF-κB or GAPDH. (B) Expression levels of p-NF-κB were quantified by densitometry and normalized with total NF-κB. Graphs show the expression levels of IL-6 (C) , TNF-α (D) , IL-1β (E) , and MCP-1 (F) by ELISA. Data are represented as the means±SD (n=6). Means with different letters are significantly different from one another (P

    Article Snippet: Antibodies to collagen I (A2), E-cadherin, TGF-βRI, CD68, EGFR, VEGF, CD31 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as well as HDAC6 siRNA were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

    Techniques: Inhibition, Activation Assay, Injection, Expressing, Enzyme-linked Immunosorbent Assay

    Human papillomavirus (HPV) E7 oncoproteins increase catalase protein levels and activity and decrease SOD1 protein levels and SOD activity. Representative immunoblot (A) and densitometric analysis (B) of catalase levels in C33A cells 48 hours after transfection with HA-tagged HPV16 E7, HIS-tagged HPV18 E7 or control pcDNA3 plasmids. Catalase enzymatic activity in cells harbouring HPV E7 oncoproteins (C). Representative immunoblot of SOD1 and SOD2 (D, F) and respective densitometric analysis (E, G) in C33A transfected cells. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. SOD enzymatic activity in C33A transfected cells is shown (H). Data is expressed as the mean±SD, Tukey's test *p

    Journal: International Journal of Biological Sciences

    Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

    doi: 10.7150/ijbs.21547

    Figure Lengend Snippet: Human papillomavirus (HPV) E7 oncoproteins increase catalase protein levels and activity and decrease SOD1 protein levels and SOD activity. Representative immunoblot (A) and densitometric analysis (B) of catalase levels in C33A cells 48 hours after transfection with HA-tagged HPV16 E7, HIS-tagged HPV18 E7 or control pcDNA3 plasmids. Catalase enzymatic activity in cells harbouring HPV E7 oncoproteins (C). Representative immunoblot of SOD1 and SOD2 (D, F) and respective densitometric analysis (E, G) in C33A transfected cells. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. SOD enzymatic activity in C33A transfected cells is shown (H). Data is expressed as the mean±SD, Tukey's test *p

    Article Snippet: Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam).

    Techniques: Activity Assay, Transfection

    Human papillomavirus (HPV) E6/E7 co-expressed oncoproteins decrease SOD1 protein levels and SOD activity. Representative immunoblot (A) and densitometric analysis (B) of catalase in C33A cells. Catalase enzymatic activity in transfected cells (C). Immunoblot and densitometric analysis of SOD1 (D, E) and SOD2 (F, G). SOD enzymatic activity (H) in C33A transfected cells. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are presented as the mean±SD, Tukey's test *p

    Journal: International Journal of Biological Sciences

    Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

    doi: 10.7150/ijbs.21547

    Figure Lengend Snippet: Human papillomavirus (HPV) E6/E7 co-expressed oncoproteins decrease SOD1 protein levels and SOD activity. Representative immunoblot (A) and densitometric analysis (B) of catalase in C33A cells. Catalase enzymatic activity in transfected cells (C). Immunoblot and densitometric analysis of SOD1 (D, E) and SOD2 (F, G). SOD enzymatic activity (H) in C33A transfected cells. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are presented as the mean±SD, Tukey's test *p

    Article Snippet: Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam).

    Techniques: Activity Assay, Transfection

    Human papillomavirus (HPV) E1, E2 and E1/E2 increase catalase activity, while SOD1 and SOD2 protein levels and SOD activity decrease. Representative immunoblot (A) and densitometric analysis (B) of catalase levels in C33 cells transfected with HA-tagged HPV18 E1, HPV18 E2, HPV 18 E1/E2 or control pcDNA3 plasmids 48 hours after transfection. Catalase enzymatic activity in C33A transfected cells (C). Representative immunoblot of SOD1 (D) and SOD2 (F) and densitometric analysis of SOD1 (E) and SOD2 (G) levels in C33 cells transfected with HA-tagged HPV18 E1, HPV18 E2, HPV 18 E1/E2 or control pcDNA3 plasmids. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. SOD enzymatic activity in C33A transfected cells (H). Data are presented as mean±SD. *p

    Journal: International Journal of Biological Sciences

    Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

    doi: 10.7150/ijbs.21547

    Figure Lengend Snippet: Human papillomavirus (HPV) E1, E2 and E1/E2 increase catalase activity, while SOD1 and SOD2 protein levels and SOD activity decrease. Representative immunoblot (A) and densitometric analysis (B) of catalase levels in C33 cells transfected with HA-tagged HPV18 E1, HPV18 E2, HPV 18 E1/E2 or control pcDNA3 plasmids 48 hours after transfection. Catalase enzymatic activity in C33A transfected cells (C). Representative immunoblot of SOD1 (D) and SOD2 (F) and densitometric analysis of SOD1 (E) and SOD2 (G) levels in C33 cells transfected with HA-tagged HPV18 E1, HPV18 E2, HPV 18 E1/E2 or control pcDNA3 plasmids. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. SOD enzymatic activity in C33A transfected cells (H). Data are presented as mean±SD. *p

    Article Snippet: Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam).

    Techniques: Activity Assay, Transfection

    Human papillomavirus (HPV) E6/E7 co-expressed oncoproteins do not affect DNA integrity in C33A cells. Immunoblot of γH2AX, H2AX (A) and densitometric analysis (B) of γH2AX in relation to H2AX. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are presented as the mean±SD, n=3.

    Journal: International Journal of Biological Sciences

    Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

    doi: 10.7150/ijbs.21547

    Figure Lengend Snippet: Human papillomavirus (HPV) E6/E7 co-expressed oncoproteins do not affect DNA integrity in C33A cells. Immunoblot of γH2AX, H2AX (A) and densitometric analysis (B) of γH2AX in relation to H2AX. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are presented as the mean±SD, n=3.

    Article Snippet: Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam).

    Techniques:

    Human papillomavirus (HPV) 18 E1/E2 promote DNA damage in C33A transfected cells. Representative immunoblot (A) and densitometric analysis (B) of γH2AX and H2AX proteins. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX at 48 hours post transfection and presented as the mean±SD, Tukey's test **p

    Journal: International Journal of Biological Sciences

    Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

    doi: 10.7150/ijbs.21547

    Figure Lengend Snippet: Human papillomavirus (HPV) 18 E1/E2 promote DNA damage in C33A transfected cells. Representative immunoblot (A) and densitometric analysis (B) of γH2AX and H2AX proteins. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX at 48 hours post transfection and presented as the mean±SD, Tukey's test **p

    Article Snippet: Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam).

    Techniques: Transfection

    Human papillomavirus (HPV) E6 oncoproteins decrease catalase protein levels and activity and do not affect SOD1/2 levels or activity. Representative immunoblot (A) and quantitative densitometric (B) showing E6 expression and catalase protein levels in C33A cells after 48 hours of transfection with Flag-tagged HPV16 E6 or HPV18 E6 and p3X vector; glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Catalase enzymatic activity in C33A transfected cells expressing HPV16 or 18 E6 oncoproteins (C). Representative immunoblot (D) and densitometric analysis (E) of superoxide dismutase (SOD) 1 in C33A transfected cells transfected; GAPDH was used as a loading control. Representative immunoblot (F) and densitometric analysis (G) of SOD2 expression, in relation to GAPDH. SOD enzymatic activity in C33A transfected cells (H). Data are expressed as the mean±SD. Tukey's test *p

    Journal: International Journal of Biological Sciences

    Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

    doi: 10.7150/ijbs.21547

    Figure Lengend Snippet: Human papillomavirus (HPV) E6 oncoproteins decrease catalase protein levels and activity and do not affect SOD1/2 levels or activity. Representative immunoblot (A) and quantitative densitometric (B) showing E6 expression and catalase protein levels in C33A cells after 48 hours of transfection with Flag-tagged HPV16 E6 or HPV18 E6 and p3X vector; glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Catalase enzymatic activity in C33A transfected cells expressing HPV16 or 18 E6 oncoproteins (C). Representative immunoblot (D) and densitometric analysis (E) of superoxide dismutase (SOD) 1 in C33A transfected cells transfected; GAPDH was used as a loading control. Representative immunoblot (F) and densitometric analysis (G) of SOD2 expression, in relation to GAPDH. SOD enzymatic activity in C33A transfected cells (H). Data are expressed as the mean±SD. Tukey's test *p

    Article Snippet: Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam).

    Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation

    Human papillomavirus (HPV) E6 oncoproteins increase DNA damage in C33A cells. Representative immunoblots (A) showing total H2AX and γH2AX in C33A cells 48 hours after transfection with Flag-tagged HPV16 E6, HPV18 E6 and p3X vector. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Densitometry of γH2AX and H2AX proteins (B). Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are shown as the mean±SD. Tukey's test *p

    Journal: International Journal of Biological Sciences

    Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

    doi: 10.7150/ijbs.21547

    Figure Lengend Snippet: Human papillomavirus (HPV) E6 oncoproteins increase DNA damage in C33A cells. Representative immunoblots (A) showing total H2AX and γH2AX in C33A cells 48 hours after transfection with Flag-tagged HPV16 E6, HPV18 E6 and p3X vector. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Densitometry of γH2AX and H2AX proteins (B). Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are shown as the mean±SD. Tukey's test *p

    Article Snippet: Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam).

    Techniques: Western Blot, Transfection, Plasmid Preparation

    Human papillomavirus (HPV) E7 oncoproteins do not alter phospho-histone 2 AX (γH2AX) protein levels. Representative immunoblot of total H2AX and γH2AX (A) and densitometric analysis (B) of the ratio γH2AX/H2AX in C33A cells 48 h post-transfection. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are presented as the mean±SD, n=3.

    Journal: International Journal of Biological Sciences

    Article Title: Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

    doi: 10.7150/ijbs.21547

    Figure Lengend Snippet: Human papillomavirus (HPV) E7 oncoproteins do not alter phospho-histone 2 AX (γH2AX) protein levels. Representative immunoblot of total H2AX and γH2AX (A) and densitometric analysis (B) of the ratio γH2AX/H2AX in C33A cells 48 h post-transfection. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as the ratio of relative units between γH2AX and H2AX. Data are presented as the mean±SD, n=3.

    Article Snippet: Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam).

    Techniques: Transfection

    Inhibition of SIRT1 expression enhances VSVΔM51 infectivity and cell death in PC-3 cells. (A and B) PC-3 cells were treated with SAHA (5 μM), RESM (5 μM), TBSA (10 μM), or MS-275 (10 μM) for 24 h or were left untreated (DMSO) and were subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ) for 24 h. (A) Total cell extracts were analyzed by immunoblotting for SIRT1, IκB α, and VSV G. β-Actin was used as a loading control. Results are from a representative experiment. (B) Analysis of Sirt1 gene expression by qPCR. Gene expression level was calculated using the ΔΔ C T method. Data represent means ± SD of results from three independent experiments. (C and D) PC-3 cells were treated with increasing concentrations of nicotinamide (NAM) for 24 h and subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ) for 24 h. (C) Infectivity was determined at 24 h p.i. by fluorescence microscopy or quantified by flow cytometry. (D) Total cell extracts were analyzed by immunoblotting for SIRT1, p21, p62/SQSTM1, and LC3B. GAPDH was used as a loading control. Results are from a representative experiment. (E to I) PC-3 cells were transfected with control (CTR) or Sirt1 siRNA and were infected 48 h later with VSVΔM51-GFP (MOI of 0.01 or 0.05). (E) Whole-cell extracts were analyzed by immunoblotting for SIRT1 and VSV G. GAPDH was used as a loading control. Results are from a representative experiment. Lipo, Lipofectamine; siCTR, small interfering control; siSIRT1, small interfering SIRT1. (F and G) Viral infectivity was determined at 24 h p.i. by fluorescence microscopy (F) and measured by flow cytometry (G). Data represent means ± SD of results from three independent experiments. (H) Viral RNA levels were measured by qPCR. (I) Cell death was assessed using 7-AAD staining by flow cytometry.

    Journal: Journal of Virology

    Article Title: SIRT1 Modulates the Sensitivity of Prostate Cancer Cells to Vesicular Stomatitis Virus Oncolysis

    doi: 10.1128/JVI.00626-19

    Figure Lengend Snippet: Inhibition of SIRT1 expression enhances VSVΔM51 infectivity and cell death in PC-3 cells. (A and B) PC-3 cells were treated with SAHA (5 μM), RESM (5 μM), TBSA (10 μM), or MS-275 (10 μM) for 24 h or were left untreated (DMSO) and were subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ) for 24 h. (A) Total cell extracts were analyzed by immunoblotting for SIRT1, IκB α, and VSV G. β-Actin was used as a loading control. Results are from a representative experiment. (B) Analysis of Sirt1 gene expression by qPCR. Gene expression level was calculated using the ΔΔ C T method. Data represent means ± SD of results from three independent experiments. (C and D) PC-3 cells were treated with increasing concentrations of nicotinamide (NAM) for 24 h and subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ) for 24 h. (C) Infectivity was determined at 24 h p.i. by fluorescence microscopy or quantified by flow cytometry. (D) Total cell extracts were analyzed by immunoblotting for SIRT1, p21, p62/SQSTM1, and LC3B. GAPDH was used as a loading control. Results are from a representative experiment. (E to I) PC-3 cells were transfected with control (CTR) or Sirt1 siRNA and were infected 48 h later with VSVΔM51-GFP (MOI of 0.01 or 0.05). (E) Whole-cell extracts were analyzed by immunoblotting for SIRT1 and VSV G. GAPDH was used as a loading control. Results are from a representative experiment. Lipo, Lipofectamine; siCTR, small interfering control; siSIRT1, small interfering SIRT1. (F and G) Viral infectivity was determined at 24 h p.i. by fluorescence microscopy (F) and measured by flow cytometry (G). Data represent means ± SD of results from three independent experiments. (H) Viral RNA levels were measured by qPCR. (I) Cell death was assessed using 7-AAD staining by flow cytometry.

    Article Snippet: Anti-VSV G, anti-SIRT1, anti-p21, anti-p62, anti-Bcl-xL, and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) were purchased from Santa Cruz Biotechnology.

    Techniques: Inhibition, Expressing, Infection, Mass Spectrometry, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Transfection, Staining

    miR-34a and SIRT1 expression levels determine the permissivity of prostate cancer cells to VSVΔM51 infection. (A) Total RNA containing miRNA from PC-3, DU-145, and LNCaP cells was extracted and subjected to real-time RT-PCR for miR-34a. The expression level of miR34a was normalized to U6. Data are expressed as means ± SD ( n = 3). (B) Total lysates from PC-3, DU-145, and LNCaP cells were analyzed by immunoblotting for SIRT1 expression. GAPDH was used as a loading control. Results are from a representative experiment. (C) PC-3, DU-145, and LNCaP cells were infected with VSVΔM51-GFP (MOI of 10 −2 ). Infectivity was determined at 24 h p.i. by fluorescence microscopy or quantified by flow cytometry. Data represent means ± SD of results from three experiments.

    Journal: Journal of Virology

    Article Title: SIRT1 Modulates the Sensitivity of Prostate Cancer Cells to Vesicular Stomatitis Virus Oncolysis

    doi: 10.1128/JVI.00626-19

    Figure Lengend Snippet: miR-34a and SIRT1 expression levels determine the permissivity of prostate cancer cells to VSVΔM51 infection. (A) Total RNA containing miRNA from PC-3, DU-145, and LNCaP cells was extracted and subjected to real-time RT-PCR for miR-34a. The expression level of miR34a was normalized to U6. Data are expressed as means ± SD ( n = 3). (B) Total lysates from PC-3, DU-145, and LNCaP cells were analyzed by immunoblotting for SIRT1 expression. GAPDH was used as a loading control. Results are from a representative experiment. (C) PC-3, DU-145, and LNCaP cells were infected with VSVΔM51-GFP (MOI of 10 −2 ). Infectivity was determined at 24 h p.i. by fluorescence microscopy or quantified by flow cytometry. Data represent means ± SD of results from three experiments.

    Article Snippet: Anti-VSV G, anti-SIRT1, anti-p21, anti-p62, anti-Bcl-xL, and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) were purchased from Santa Cruz Biotechnology.

    Techniques: Expressing, Infection, Quantitative RT-PCR, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    Resveratrol-mediated SIRT1 activation reverses the synergistic effect of SAHA on VSVΔM51 replication. (A) PC-3 cells were treated with resveratrol (Rsv) at different concentrations for 2 h prior to the addition of SAHA (5 μM) or not (DMSO) for 24 h and were subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ). Infectivity was determined at 24 h p.i. by fluorescence microscopy (A) or quantified by flow cytometry (B). Data represent means ± SD of results from three experiments. (C) Total lysates were analyzed by immunoblotting for SIRT1 expression. GAPDH was used as a loading control. Results are from a representative experiment. (D) DU-145 cells were treated with Rsv 50 μM or not for 24 h, and subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ). Infectivity was determined at 24 h p.i. by fluorescence microscopy or quantified by flow cytometry. Data represent means ± SD of results from three experiments. (E to H) U-2 OS cells were transfected with SIRT1 WT, H363Y mutant or the empty vector (vec) and 24 h later were infected with VSVΔM51-GFP (MOI of 10 −3 or 10 −2 ). Viral infectivity was determined at 24 h p.i. by fluorescence microscopy (E) and measured by flow cytometry as the percentage of GFP-positive cells (F) and GFP mean fluorescence intensity (MFI) (G). (H) Whole-cell extracts were analyzed by immunoblotting for FLAG, SIRT1, and VSV G. L.E., long exposure; S.E., short exposure. GAPDH was used as a loading control. Results are from a representative experiment. Data represent means ± SD of results from three independent experiments.

    Journal: Journal of Virology

    Article Title: SIRT1 Modulates the Sensitivity of Prostate Cancer Cells to Vesicular Stomatitis Virus Oncolysis

    doi: 10.1128/JVI.00626-19

    Figure Lengend Snippet: Resveratrol-mediated SIRT1 activation reverses the synergistic effect of SAHA on VSVΔM51 replication. (A) PC-3 cells were treated with resveratrol (Rsv) at different concentrations for 2 h prior to the addition of SAHA (5 μM) or not (DMSO) for 24 h and were subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ). Infectivity was determined at 24 h p.i. by fluorescence microscopy (A) or quantified by flow cytometry (B). Data represent means ± SD of results from three experiments. (C) Total lysates were analyzed by immunoblotting for SIRT1 expression. GAPDH was used as a loading control. Results are from a representative experiment. (D) DU-145 cells were treated with Rsv 50 μM or not for 24 h, and subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ). Infectivity was determined at 24 h p.i. by fluorescence microscopy or quantified by flow cytometry. Data represent means ± SD of results from three experiments. (E to H) U-2 OS cells were transfected with SIRT1 WT, H363Y mutant or the empty vector (vec) and 24 h later were infected with VSVΔM51-GFP (MOI of 10 −3 or 10 −2 ). Viral infectivity was determined at 24 h p.i. by fluorescence microscopy (E) and measured by flow cytometry as the percentage of GFP-positive cells (F) and GFP mean fluorescence intensity (MFI) (G). (H) Whole-cell extracts were analyzed by immunoblotting for FLAG, SIRT1, and VSV G. L.E., long exposure; S.E., short exposure. GAPDH was used as a loading control. Results are from a representative experiment. Data represent means ± SD of results from three independent experiments.

    Article Snippet: Anti-VSV G, anti-SIRT1, anti-p21, anti-p62, anti-Bcl-xL, and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) were purchased from Santa Cruz Biotechnology.

    Techniques: Activation Assay, Infection, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Expressing, Transfection, Mutagenesis, Plasmid Preparation

    Inhibition mediated by HDAC1 and HDAC3 induces a marked depletion of S-phase cells and increase in the levels of G 2 /M cells in PC-3 cell cycle arrest. (A) The cell cycle distribution of PC-3 cells treated for 24 h with increasing concentrations of SAHA (0 to 5 μM) was analyzed by propidium iodide (PI) staining by flow cytometry. The percentages of cells in the G 0 /G 1 , S, and G 2 /M phases were evaluated by measuring DNA content. Data represent means ± SD of results from three independent experiments. (B and C) PC-3 cells, pretreated as described for panel A, were subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ). Infectivity was quantified at 24 h postinfection (p.i.) by flow cytometry. (B) Cell death was assessed using annexin V (AnnV)/7AAD staining by flow cytometry. (C) Data represent means ± SD of results from three experiments. (D) PC-3 cells treated with SAHA (5 μM) for 24 h (T0), as well as PC-3 cells pretreated with SAHA and then infected with VSVΔM51-GFP (MOI of 10 −2 ) for a subsequent 24 h, were analyzed for CDKN1A gene expression by qPCR. Gene expression levels were calculated using the threshold cycle (ΔΔ C T ) method. Data represent means ± SD of results from three independent experiments. The same samples were used to obtain total cell extracts and analyzed by immunoblotting for p21 protein levels. β-Actin was used as a loading control. Results are from a representative experiment. F.I., fold increase. (E to G) PC-3 cells were treated with SAHA (5 μM), RESM (5 μM), TBSA (10 μM), or MS-275 (10 μM) for 24 h (T0) or were left untreated (DMSO). (E) The cell cycle distribution was analyzed by PI staining by using flow cytometer. (F) CDKN1A, CDK6, and CCDN1 gene expression levels were evaluated by qPCR analysis. The gene expression levels were calculated using the ΔΔ C T method. Data are representative of results from three independent experiments. (G) Total cell extracts were analyzed by immunoblotting for p21, p16INK4A, IκB α, SQSTM1, and LC3B. GAPDH was used as a loading control. Acetyl-α-tubulin and acetyl-histone H3 were used as controls for analysis of the specific activity of the different HDAC inhibitors. Results are from a representative experiment.

    Journal: Journal of Virology

    Article Title: SIRT1 Modulates the Sensitivity of Prostate Cancer Cells to Vesicular Stomatitis Virus Oncolysis

    doi: 10.1128/JVI.00626-19

    Figure Lengend Snippet: Inhibition mediated by HDAC1 and HDAC3 induces a marked depletion of S-phase cells and increase in the levels of G 2 /M cells in PC-3 cell cycle arrest. (A) The cell cycle distribution of PC-3 cells treated for 24 h with increasing concentrations of SAHA (0 to 5 μM) was analyzed by propidium iodide (PI) staining by flow cytometry. The percentages of cells in the G 0 /G 1 , S, and G 2 /M phases were evaluated by measuring DNA content. Data represent means ± SD of results from three independent experiments. (B and C) PC-3 cells, pretreated as described for panel A, were subsequently infected with VSVΔM51-GFP (MOI of 10 −2 ). Infectivity was quantified at 24 h postinfection (p.i.) by flow cytometry. (B) Cell death was assessed using annexin V (AnnV)/7AAD staining by flow cytometry. (C) Data represent means ± SD of results from three experiments. (D) PC-3 cells treated with SAHA (5 μM) for 24 h (T0), as well as PC-3 cells pretreated with SAHA and then infected with VSVΔM51-GFP (MOI of 10 −2 ) for a subsequent 24 h, were analyzed for CDKN1A gene expression by qPCR. Gene expression levels were calculated using the threshold cycle (ΔΔ C T ) method. Data represent means ± SD of results from three independent experiments. The same samples were used to obtain total cell extracts and analyzed by immunoblotting for p21 protein levels. β-Actin was used as a loading control. Results are from a representative experiment. F.I., fold increase. (E to G) PC-3 cells were treated with SAHA (5 μM), RESM (5 μM), TBSA (10 μM), or MS-275 (10 μM) for 24 h (T0) or were left untreated (DMSO). (E) The cell cycle distribution was analyzed by PI staining by using flow cytometer. (F) CDKN1A, CDK6, and CCDN1 gene expression levels were evaluated by qPCR analysis. The gene expression levels were calculated using the ΔΔ C T method. Data are representative of results from three independent experiments. (G) Total cell extracts were analyzed by immunoblotting for p21, p16INK4A, IκB α, SQSTM1, and LC3B. GAPDH was used as a loading control. Acetyl-α-tubulin and acetyl-histone H3 were used as controls for analysis of the specific activity of the different HDAC inhibitors. Results are from a representative experiment.

    Article Snippet: Anti-VSV G, anti-SIRT1, anti-p21, anti-p62, anti-Bcl-xL, and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) were purchased from Santa Cruz Biotechnology.

    Techniques: Inhibition, Staining, Flow Cytometry, Cytometry, Infection, Expressing, Real-time Polymerase Chain Reaction, Mass Spectrometry, Activity Assay

    UA sensitizes HepG2/DDP cells to low-dose cisplatin via inhibition of Nrf2/ARE signaling pathway. Notes: ( A ) HepG2/DDP cells were transfected with Nrf2 siRNA (si-Nrf2) or negative control (si-Con), or ( C ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with 8.92 μg/mL cisplatin (IC 30 of cisplatin for HepG2/DDP cells) and/or UA (2.25 μg/mL) for 48 hours. The level of Nrf2, HO-1, NQO1, and GST was detected by Western blot analysis. ( B ) HepG2/DDP cells were transfected with si-Nrf2 or si-Con, or ( D ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with series concentration of cisplatin (2–512 μg/mL) and/or UA (2.25 μg/mL) for 48 hours. The inhibition rate of cell was detected by CCK8 assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: ARE, antioxidant response element; CCK8, Cell Counting Kit 8; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin–resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; IC 30 , 30% inhibitory concentration; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; siRNA, small interfering RNA; UA, ursolic acid.

    Journal: Drug Design, Development and Therapy

    Article Title: Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

    doi: 10.2147/DDDT.S110505

    Figure Lengend Snippet: UA sensitizes HepG2/DDP cells to low-dose cisplatin via inhibition of Nrf2/ARE signaling pathway. Notes: ( A ) HepG2/DDP cells were transfected with Nrf2 siRNA (si-Nrf2) or negative control (si-Con), or ( C ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with 8.92 μg/mL cisplatin (IC 30 of cisplatin for HepG2/DDP cells) and/or UA (2.25 μg/mL) for 48 hours. The level of Nrf2, HO-1, NQO1, and GST was detected by Western blot analysis. ( B ) HepG2/DDP cells were transfected with si-Nrf2 or si-Con, or ( D ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with series concentration of cisplatin (2–512 μg/mL) and/or UA (2.25 μg/mL) for 48 hours. The inhibition rate of cell was detected by CCK8 assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: ARE, antioxidant response element; CCK8, Cell Counting Kit 8; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin–resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; IC 30 , 30% inhibitory concentration; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; siRNA, small interfering RNA; UA, ursolic acid.

    Article Snippet: Anti-Nrf2, anti-HO-1, anti-NQO1, anti-GST, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, and horseradish-peroxidase-conjugated secondary antibody were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

    Techniques: Inhibition, Transfection, Negative Control, Plasmid Preparation, Western Blot, Concentration Assay, CCK-8 Assay, Cell Counting, Standard Deviation, Small Interfering RNA

    Nrf2 was overexpressed in cisplatin-resistant human hepatocellular carcinoma HepG2/DDP cells. Notes: ( A ) HepG2 cells were treated with series concentration of cisplatin (0.1–25.6 μg/mL) for 48 hours. ( B ) HepG2/DDP cells were treated with series concentration of cisplatin (2–512 μg/mL) for 48 hours. ( C ) The level of Nrf2 and its downstream target genes HO-1, NQO1, and GST in HepG2 and HepG2/DDP cells was detected by Western blot assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2, hepatocellular carcinoma cell line; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation.

    Journal: Drug Design, Development and Therapy

    Article Title: Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

    doi: 10.2147/DDDT.S110505

    Figure Lengend Snippet: Nrf2 was overexpressed in cisplatin-resistant human hepatocellular carcinoma HepG2/DDP cells. Notes: ( A ) HepG2 cells were treated with series concentration of cisplatin (0.1–25.6 μg/mL) for 48 hours. ( B ) HepG2/DDP cells were treated with series concentration of cisplatin (2–512 μg/mL) for 48 hours. ( C ) The level of Nrf2 and its downstream target genes HO-1, NQO1, and GST in HepG2 and HepG2/DDP cells was detected by Western blot assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2, hepatocellular carcinoma cell line; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation.

    Article Snippet: Anti-Nrf2, anti-HO-1, anti-NQO1, anti-GST, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, and horseradish-peroxidase-conjugated secondary antibody were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

    Techniques: Concentration Assay, Western Blot, Standard Deviation

    UA–cisplatin combination downregulates Nrf2 and its substrates. Notes: The protein expression levels of Nrf2, HO-1, NQO1, and GST of HepG2/DDP cells treated with 8.92 μg/mL cisplatin and/or UA (2.25 μg/mL) for 48 hours were detected by Western blot analysis. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; UA, ursolic acid.

    Journal: Drug Design, Development and Therapy

    Article Title: Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

    doi: 10.2147/DDDT.S110505

    Figure Lengend Snippet: UA–cisplatin combination downregulates Nrf2 and its substrates. Notes: The protein expression levels of Nrf2, HO-1, NQO1, and GST of HepG2/DDP cells treated with 8.92 μg/mL cisplatin and/or UA (2.25 μg/mL) for 48 hours were detected by Western blot analysis. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; UA, ursolic acid.

    Article Snippet: Anti-Nrf2, anti-HO-1, anti-NQO1, anti-GST, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, and horseradish-peroxidase-conjugated secondary antibody were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

    Techniques: Expressing, Western Blot, Standard Deviation

    Effects of sodium ferulate on cycloxygenase-2, prostaglandin E2, inducible nitric oxide synthase, and nitric oxide . Effects of sodium ferulate (SF) on cycloxygenase-2 (COX-2), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) expression and synthesis of rat osteoarthritis (OA) chondrocytes induced by IL-1β. (a) Effects of SF on protein expression of COX-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat OA chondrocytes. Chondrocytes were incubated with SF alone for 24 hours, and then co-treated with IL-1β and SF for 48 hours. Expression of COX-2 and GAPDH determined by western blotting. (b) Effects of SF on gene expression of iNOS and GAPDH in rat OA chondrocytes. Level of iNOS was evaluated by real-time quantitative PCR. (c) Effects of SF on NO synthesis of rat OA chondrocytes induced by IL-1β. NO production was measured as nitrite (NO 2 - ) in the culture medium by the Griess reaction. NO concentration was calculated (expressed in μmol/l). (d) Effects of SF on expression of PGE2 in rat OA chondrocytes. The release of PGE2 was analyzed by ELISA and the concentration was calculated (expressed in pg/ml). Values represent mean ± standard error of the mean of three different simples. ** P

    Journal: Arthritis Research & Therapy

    Article Title: TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1?-induced rat osteoarthritis chondrocytes in vitro

    doi: 10.1186/ar4085

    Figure Lengend Snippet: Effects of sodium ferulate on cycloxygenase-2, prostaglandin E2, inducible nitric oxide synthase, and nitric oxide . Effects of sodium ferulate (SF) on cycloxygenase-2 (COX-2), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) expression and synthesis of rat osteoarthritis (OA) chondrocytes induced by IL-1β. (a) Effects of SF on protein expression of COX-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat OA chondrocytes. Chondrocytes were incubated with SF alone for 24 hours, and then co-treated with IL-1β and SF for 48 hours. Expression of COX-2 and GAPDH determined by western blotting. (b) Effects of SF on gene expression of iNOS and GAPDH in rat OA chondrocytes. Level of iNOS was evaluated by real-time quantitative PCR. (c) Effects of SF on NO synthesis of rat OA chondrocytes induced by IL-1β. NO production was measured as nitrite (NO 2 - ) in the culture medium by the Griess reaction. NO concentration was calculated (expressed in μmol/l). (d) Effects of SF on expression of PGE2 in rat OA chondrocytes. The release of PGE2 was analyzed by ELISA and the concentration was calculated (expressed in pg/ml). Values represent mean ± standard error of the mean of three different simples. ** P

    Article Snippet: Rabbit anti-TNFR-1 polyclonal antibody, anti-TNF receptor-associated death domain (anti-TRADD), anti-TRAF-2, anti-IKKα, anti-phospho-IKKα, anti-IKKβ, anti-phospho-IKKβ, anti-IκBα, anti-phospho-IκBα, anti-caspase-3, anti-COX-2, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase-conjugated anti-rabbit were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Effects of sodium ferulate on the caspase cascade apoptosis pathway in rat osteoarthritis chondrocytes . Effects of sodium ferulate (SF) on the TNF/TNF receptor (TNFR)-mediated caspase cascade apoptosis pathway of rat osteoarthritis (OA) chondrocytes induced by IL-1β. (a) Effects of SF on expression of TNFα in rat OA chondrocytes. Chondrocytes were incubated with SF alone for 24 hours, and then co-treated with IL-1β and SF for 48 hours. Supernatants were collected after 72 hours. Release of TNFα was analyzed by ELISA. (b) Effects of SF on protein expression of TNFR-1, TNF receptor-associated death domain (TRADD), caspase-8, caspase-3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat OA chondrocytes. Expression of proteins determined by western blotting. (c) Effects of SF on activity of caspase-8 and caspase-3 in rat OA chondrocytes. An activity unit was defined as the amount of enzyme that will cleave 1.0 nmol colorimetric substrate (acetyl-Asp-Glu-Val-Asp p -nitroanilide or acetyl-Ile-Glu-Thr-Asp p -nitroanilide) per hour at 37°C under saturated substrate concentrations. Caspase activity was expressed as activity units compared with total protein content (unit/μg pro). (d, e, f, g) Relative level of TNFR-1, TRADD, caspase-8 and caspase-3 normalized to GAPDH and compared with normal control, quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak, Rochester, NY, USA) and expressed as mean optical density. Values represent mean ± standard error of the mean of three different simples. ** P

    Journal: Arthritis Research & Therapy

    Article Title: TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1?-induced rat osteoarthritis chondrocytes in vitro

    doi: 10.1186/ar4085

    Figure Lengend Snippet: Effects of sodium ferulate on the caspase cascade apoptosis pathway in rat osteoarthritis chondrocytes . Effects of sodium ferulate (SF) on the TNF/TNF receptor (TNFR)-mediated caspase cascade apoptosis pathway of rat osteoarthritis (OA) chondrocytes induced by IL-1β. (a) Effects of SF on expression of TNFα in rat OA chondrocytes. Chondrocytes were incubated with SF alone for 24 hours, and then co-treated with IL-1β and SF for 48 hours. Supernatants were collected after 72 hours. Release of TNFα was analyzed by ELISA. (b) Effects of SF on protein expression of TNFR-1, TNF receptor-associated death domain (TRADD), caspase-8, caspase-3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat OA chondrocytes. Expression of proteins determined by western blotting. (c) Effects of SF on activity of caspase-8 and caspase-3 in rat OA chondrocytes. An activity unit was defined as the amount of enzyme that will cleave 1.0 nmol colorimetric substrate (acetyl-Asp-Glu-Val-Asp p -nitroanilide or acetyl-Ile-Glu-Thr-Asp p -nitroanilide) per hour at 37°C under saturated substrate concentrations. Caspase activity was expressed as activity units compared with total protein content (unit/μg pro). (d, e, f, g) Relative level of TNFR-1, TRADD, caspase-8 and caspase-3 normalized to GAPDH and compared with normal control, quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak, Rochester, NY, USA) and expressed as mean optical density. Values represent mean ± standard error of the mean of three different simples. ** P

    Article Snippet: Rabbit anti-TNFR-1 polyclonal antibody, anti-TNF receptor-associated death domain (anti-TRADD), anti-TRAF-2, anti-IKKα, anti-phospho-IKKα, anti-IKKβ, anti-phospho-IKKβ, anti-IκBα, anti-phospho-IκBα, anti-caspase-3, anti-COX-2, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase-conjugated anti-rabbit were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Software

    Effects of sodium ferulate on the IKK/NF-κB signaling pathway of rat osteoarthritis chondrocytes . Effects of sodium ferulate (SF) on the TNF/TNF receptor (TNFR)-mediated IKK/NF-κB signaling pathway of rat osteoarthritis (OA) chondrocytes induced by IL-1β. (a) Effects of SF on protein expression of TNF receptor-associated factor 2 (TRAF-2), inhibitor of NF-κB kinase (IKK) subunit alpha (IKKα), IKK subunit beta (IKKβ), NF-κB light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα), phosphorylation of IKKα (p-IKKα), phosphorylation of IKKβ (p-IKKβ), phosphorylation of IκBα (p-IκBα) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat OA chondrocytes. Chondrocytes were incubated with SF alone for 24 hours, and then co-treated with IL-1β and SF for 48 hours. Expressions of proteins determined by western blotting. (b, c, d, e) Relative level of TRAF-2, IKKα, IKKβ, IκBα, p-IKKα, p-IKKβ and p-IκBα normalized to GAPDH and compared with normal control, quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak, Rochester, NY, USA) and expressed as mean optical density. Values represent mean ± standard error of the mean of three different simples. ** P

    Journal: Arthritis Research & Therapy

    Article Title: TNF/TNFR signal transduction pathway-mediated anti-apoptosis and anti-inflammatory effects of sodium ferulate on IL-1?-induced rat osteoarthritis chondrocytes in vitro

    doi: 10.1186/ar4085

    Figure Lengend Snippet: Effects of sodium ferulate on the IKK/NF-κB signaling pathway of rat osteoarthritis chondrocytes . Effects of sodium ferulate (SF) on the TNF/TNF receptor (TNFR)-mediated IKK/NF-κB signaling pathway of rat osteoarthritis (OA) chondrocytes induced by IL-1β. (a) Effects of SF on protein expression of TNF receptor-associated factor 2 (TRAF-2), inhibitor of NF-κB kinase (IKK) subunit alpha (IKKα), IKK subunit beta (IKKβ), NF-κB light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα), phosphorylation of IKKα (p-IKKα), phosphorylation of IKKβ (p-IKKβ), phosphorylation of IκBα (p-IκBα) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat OA chondrocytes. Chondrocytes were incubated with SF alone for 24 hours, and then co-treated with IL-1β and SF for 48 hours. Expressions of proteins determined by western blotting. (b, c, d, e) Relative level of TRAF-2, IKKα, IKKβ, IκBα, p-IKKα, p-IKKβ and p-IκBα normalized to GAPDH and compared with normal control, quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak, Rochester, NY, USA) and expressed as mean optical density. Values represent mean ± standard error of the mean of three different simples. ** P

    Article Snippet: Rabbit anti-TNFR-1 polyclonal antibody, anti-TNF receptor-associated death domain (anti-TRADD), anti-TRAF-2, anti-IKKα, anti-phospho-IKKα, anti-IKKβ, anti-phospho-IKKβ, anti-IκBα, anti-phospho-IκBα, anti-caspase-3, anti-COX-2, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase-conjugated anti-rabbit were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Incubation, Western Blot, Software

    Effects of BHMC on β-PIX, MMP-9, MT1-MMP, protein expression. MDA-MB-231 cells were treated with indicated concentrations of BHMC for 24 h. Cell lysates were probed with ( A ) β-PIX, ( B ) MMP-9, and ( C ) MT1-MMP and normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level. All data are the mean ± S.E.M of three independent experiments. * p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: The Molecular Targets and Anti-Invasive Effects of 2,6-bis-(4-hydroxyl-3methoxybenzylidine) cyclohexanone or BHMC in MDA-MB-231 Human Breast Cancer Cells

    doi: 10.3390/molecules23040865

    Figure Lengend Snippet: Effects of BHMC on β-PIX, MMP-9, MT1-MMP, protein expression. MDA-MB-231 cells were treated with indicated concentrations of BHMC for 24 h. Cell lysates were probed with ( A ) β-PIX, ( B ) MMP-9, and ( C ) MT1-MMP and normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level. All data are the mean ± S.E.M of three independent experiments. * p

    Article Snippet: HRP-conjugated goat anti-rabbit Ig-G and anti- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Multiple Displacement Amplification

    Inhibitory effect of curcumin on VEGF expression disappears via activating PI3K/Akt signaling pathway in neonatal rats with brain HI damage (n = 12 per group). (A) Band intensities from western blots were calculated. (B) The protein expression of p-PI3K, PI3K, p-Akt, Akt, and VEGF in the sham, HI, HI + CUR and HI + CUR + IGF-1 groups by western blotting. HI, hypoxic-ischemic injury; CUR, curcumin; IGF-1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **p

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Curcumin targets vascular endothelial growth factor via activating the PI3K/Akt signaling pathway and improves brain hypoxic-ischemic injury in neonatal rats

    doi: 10.4196/kjpp.2020.24.5.423

    Figure Lengend Snippet: Inhibitory effect of curcumin on VEGF expression disappears via activating PI3K/Akt signaling pathway in neonatal rats with brain HI damage (n = 12 per group). (A) Band intensities from western blots were calculated. (B) The protein expression of p-PI3K, PI3K, p-Akt, Akt, and VEGF in the sham, HI, HI + CUR and HI + CUR + IGF-1 groups by western blotting. HI, hypoxic-ischemic injury; CUR, curcumin; IGF-1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **p

    Article Snippet: Then protein samples were separated on SDS-PAGE gel, transferred to polyvinylidene fluoride membranes, and blocked with 5% nonfat milk for 1 h. Next, the membrane was incubated with anti-rat p-PI3K, PI3K, p-Akt, Akt, VEGF, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:500, Cat: sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, washed with PBS, and incubated with secondary antibody (1:5000, Cat:111-035-003; Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature.

    Techniques: Expressing, Western Blot

    Curcumin promotes the activation of PI3K/Akt signaling pathway and VEGF expression in neonatal rats with brain HI damage (n = 12 per group). (A) Band intensities from western blots were calculated. (B) The protein expression of p-PI3K, PI3K, p-Akt, Akt, and VEGF in the sham, HI, and HI + CUR groups by western blotting. HI, hypoxic-ischemic injury; CUR, curcumin; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **p

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Curcumin targets vascular endothelial growth factor via activating the PI3K/Akt signaling pathway and improves brain hypoxic-ischemic injury in neonatal rats

    doi: 10.4196/kjpp.2020.24.5.423

    Figure Lengend Snippet: Curcumin promotes the activation of PI3K/Akt signaling pathway and VEGF expression in neonatal rats with brain HI damage (n = 12 per group). (A) Band intensities from western blots were calculated. (B) The protein expression of p-PI3K, PI3K, p-Akt, Akt, and VEGF in the sham, HI, and HI + CUR groups by western blotting. HI, hypoxic-ischemic injury; CUR, curcumin; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **p

    Article Snippet: Then protein samples were separated on SDS-PAGE gel, transferred to polyvinylidene fluoride membranes, and blocked with 5% nonfat milk for 1 h. Next, the membrane was incubated with anti-rat p-PI3K, PI3K, p-Akt, Akt, VEGF, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:500, Cat: sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, washed with PBS, and incubated with secondary antibody (1:5000, Cat:111-035-003; Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature.

    Techniques: Activation Assay, Expressing, Western Blot

    Inhibition of ER stress-related apoptosis in the sciatic nerve of DPN rats by intrathecal injection of IRE1α siRNA. ( A ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against P-IRE1α and XBP-1s. β-actin was probed as loading control. The intensity is expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ( B ) TUNEL, p-JNK, and Caspae-12 were effectively eliminated as measured by immunohistochemistry method. Data are expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with Tamhane’s T2 analysis or unpaired Student’s t-test. ( C ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against GRP78, CHOP, Bcl-2, Bax and Cleaved-Caspase-3. β-actin was probed as loading control. Results of are expressed as mean ± SEM indicated as percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis (GRP78, Cleaved-Caspase-3, CHOP) or Tamhane’s T2 analysis (Bcl-2, Bax) or unpaired Student’s t-test. Δ P

    Journal: Scientific Reports

    Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

    doi: 10.1038/s41598-018-20950-9

    Figure Lengend Snippet: Inhibition of ER stress-related apoptosis in the sciatic nerve of DPN rats by intrathecal injection of IRE1α siRNA. ( A ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against P-IRE1α and XBP-1s. β-actin was probed as loading control. The intensity is expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis or unpaired Student’s t-test. ( B ) TUNEL, p-JNK, and Caspae-12 were effectively eliminated as measured by immunohistochemistry method. Data are expressed as mean ± SEM of the percentage of the respective controls and analyzed using one-way ANOVA with Tamhane’s T2 analysis or unpaired Student’s t-test. ( C ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against GRP78, CHOP, Bcl-2, Bax and Cleaved-Caspase-3. β-actin was probed as loading control. Results of are expressed as mean ± SEM indicated as percentage of the respective controls and analyzed using one-way ANOVA with LSD analysis (GRP78, Cleaved-Caspase-3, CHOP) or Tamhane’s T2 analysis (Bcl-2, Bax) or unpaired Student’s t-test. Δ P

    Article Snippet: The primary antibodies were as follows: mouse anti-IRE1α (sc-390960; 1: 1000; Santa Cruz), rabbit anti-P-IRE1α (ab48187; 1: 2000; abcam), mouse anti-XBP1 (sc-8015; 1: 1000; Abcam), mouse anti-GRP78 (sc-376768; 1: 1000; Santa Cruz), mouse anti-GADD153 (sc-7351; 1: 500; Santa Cruz), rabbit anti-Caspase-3 (ab13847; 1: 1000; abcam), rabbit anti-Caspase-12 (om273459; 1: 1000; Omnimabs), mouse anti-Bcl-2 (sc-7382; 1: 1000; Santa Cruz), mouse anti-Bax (sc-7480; 1: 1000; Santa Cruz), mouse anti-p-JNK (sc-6254; 1: 1000; Santa Cruz), and rabbit anti-PGP9.5 (ab108986; 1: 2000; Abcam).

    Techniques: Inhibition, Injection, Western Blot, TUNEL Assay, Immunohistochemistry

    Expression of CHOP, Bcl-2, Bax, p-JNK, Caspase-12 and cleaved-Caspase-3 in RSC96 cells after IRE1α siRNA transfection and exposure to high glucose. ( A ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against CHOP, Bcl-2, Bax, p-JNK, Caspase-12 and cleaved-Caspase-3. ( B ) The band intensity expressed as mean ± SEM of the percentage of the respective 25 mM glucose-treated cells and analyzed using one-way ANOVA with LSD analysis (CHOP, Bcl-2/48 h, Bax/48 h, p-JNK/24 h, Caspase-12, cleaved-Caspase-3,) or Tamhane’s T2 analysis (p-JNK/48 h, Bcl-2/24 h, Bax/24 h, Caspase-3/24 h,). Δ P

    Journal: Scientific Reports

    Article Title: IRE1α siRNA relieves endoplasmic reticulum stress-induced apoptosis and alleviates diabetic peripheral neuropathy in vivo and in vitro

    doi: 10.1038/s41598-018-20950-9

    Figure Lengend Snippet: Expression of CHOP, Bcl-2, Bax, p-JNK, Caspase-12 and cleaved-Caspase-3 in RSC96 cells after IRE1α siRNA transfection and exposure to high glucose. ( A ) Representative Western blots using tissue extracts from the sciatic nerve and probed with antibodies against CHOP, Bcl-2, Bax, p-JNK, Caspase-12 and cleaved-Caspase-3. ( B ) The band intensity expressed as mean ± SEM of the percentage of the respective 25 mM glucose-treated cells and analyzed using one-way ANOVA with LSD analysis (CHOP, Bcl-2/48 h, Bax/48 h, p-JNK/24 h, Caspase-12, cleaved-Caspase-3,) or Tamhane’s T2 analysis (p-JNK/48 h, Bcl-2/24 h, Bax/24 h, Caspase-3/24 h,). Δ P

    Article Snippet: The primary antibodies were as follows: mouse anti-IRE1α (sc-390960; 1: 1000; Santa Cruz), rabbit anti-P-IRE1α (ab48187; 1: 2000; abcam), mouse anti-XBP1 (sc-8015; 1: 1000; Abcam), mouse anti-GRP78 (sc-376768; 1: 1000; Santa Cruz), mouse anti-GADD153 (sc-7351; 1: 500; Santa Cruz), rabbit anti-Caspase-3 (ab13847; 1: 1000; abcam), rabbit anti-Caspase-12 (om273459; 1: 1000; Omnimabs), mouse anti-Bcl-2 (sc-7382; 1: 1000; Santa Cruz), mouse anti-Bax (sc-7480; 1: 1000; Santa Cruz), mouse anti-p-JNK (sc-6254; 1: 1000; Santa Cruz), and rabbit anti-PGP9.5 (ab108986; 1: 2000; Abcam).

    Techniques: Expressing, Transfection, Western Blot

    Increased expression of cAMP as well as activated Bax and caspase-3 in astrocytes of the glial lamina in glaucomatous DBA2/J mice a and b Immunohistochemical analyses of cAMP (green), activated Bax (yellow), and caspase-3 (green) as well as GFAP (red) immunoreactivity in the ONH astrocytes in the glial lamina of age-matched non-glaucomatous DBA/2J- Gpnmb + and 10-month-old glaucomatous DBA/2 J mice. Note that representative images showed increases of cAMP, activated Bax, and caspase-3 immunoreactivity in astrocytes of the glial lamina in glaucomatous DBA/2 J mice compared with non-glaucomatous DBA/2J- Gpnmb + . Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. c Quantitative analysis showed significant increases of cAMP and GFAP, as well as activated Bax and caspase-3 immunoreactivity in glaucomatous DBA/2 J mice. For each determination, the immunoreactivity in controls was normalized to a value of 1.0. Data are shown as the mean ± S.D. ( n = 5). * P

    Journal: Cell Death & Disease

    Article Title: Elevated intracellular cAMP exacerbates vulnerability to oxidative stress in optic nerve head astrocytes

    doi: 10.1038/s41419-017-0171-8

    Figure Lengend Snippet: Increased expression of cAMP as well as activated Bax and caspase-3 in astrocytes of the glial lamina in glaucomatous DBA2/J mice a and b Immunohistochemical analyses of cAMP (green), activated Bax (yellow), and caspase-3 (green) as well as GFAP (red) immunoreactivity in the ONH astrocytes in the glial lamina of age-matched non-glaucomatous DBA/2J- Gpnmb + and 10-month-old glaucomatous DBA/2 J mice. Note that representative images showed increases of cAMP, activated Bax, and caspase-3 immunoreactivity in astrocytes of the glial lamina in glaucomatous DBA/2 J mice compared with non-glaucomatous DBA/2J- Gpnmb + . Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. c Quantitative analysis showed significant increases of cAMP and GFAP, as well as activated Bax and caspase-3 immunoreactivity in glaucomatous DBA/2 J mice. For each determination, the immunoreactivity in controls was normalized to a value of 1.0. Data are shown as the mean ± S.D. ( n = 5). * P

    Article Snippet: The primary antibodies were mouse monoclonal anti-cAMP antibody (1:500; Sigma), mouse monoclonal anti-Bax antibody (6A7; 1:100; Santa Cruz), and rabbit polyclonal caspase-3 (1:100; Cell signaling).

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining

    PKA inhibition protects ONH astrocytes against oxidative stress a Cell viability analyses using MTT assay in H 2 O 2 (50 μM for 1 h)-induced ONH astrocytes treated with Rp-cAMP (50 μM). b Cell viability analysis using MTT assay in H 2 O 2 (50 μM)-induced ONH astrocytes transduced with AAV2/5-tdTomato or mCherry-PKI. Representative images showed tdTomato (red) or mCherry-PKI (red) expression in ONH astrocytes. Nuclei (blue) were stained by Hoechst 33342. Note that PKI overexpression promoted ONH astrocyte survival. Scale bars, 20 μm. c Immunoblot analyses of pAKT, total AKT, Bim and activated Bax in H 2 O 2 (50 μM for 1 h)-induced ONH astrocytes transduced with AAV2/5-tdTomato or mCherry-PKI. d A hypothetical model for the role of tmAC activation-mediated cAMP/PKA signaling pathway in ONH astrocytes against glaucomatous insults such as elevated IOP and oxidative stress. For each determination, the cell viability as well as mRNA and protein expression in controls were normalized to a value of 1.0. Data are shown as the mean ± S.D. ( n = 3). * P

    Journal: Cell Death & Disease

    Article Title: Elevated intracellular cAMP exacerbates vulnerability to oxidative stress in optic nerve head astrocytes

    doi: 10.1038/s41419-017-0171-8

    Figure Lengend Snippet: PKA inhibition protects ONH astrocytes against oxidative stress a Cell viability analyses using MTT assay in H 2 O 2 (50 μM for 1 h)-induced ONH astrocytes treated with Rp-cAMP (50 μM). b Cell viability analysis using MTT assay in H 2 O 2 (50 μM)-induced ONH astrocytes transduced with AAV2/5-tdTomato or mCherry-PKI. Representative images showed tdTomato (red) or mCherry-PKI (red) expression in ONH astrocytes. Nuclei (blue) were stained by Hoechst 33342. Note that PKI overexpression promoted ONH astrocyte survival. Scale bars, 20 μm. c Immunoblot analyses of pAKT, total AKT, Bim and activated Bax in H 2 O 2 (50 μM for 1 h)-induced ONH astrocytes transduced with AAV2/5-tdTomato or mCherry-PKI. d A hypothetical model for the role of tmAC activation-mediated cAMP/PKA signaling pathway in ONH astrocytes against glaucomatous insults such as elevated IOP and oxidative stress. For each determination, the cell viability as well as mRNA and protein expression in controls were normalized to a value of 1.0. Data are shown as the mean ± S.D. ( n = 3). * P

    Article Snippet: The primary antibodies were mouse monoclonal anti-cAMP antibody (1:500; Sigma), mouse monoclonal anti-Bax antibody (6A7; 1:100; Santa Cruz), and rabbit polyclonal caspase-3 (1:100; Cell signaling).

    Techniques: Inhibition, MTT Assay, Transduction, Expressing, Staining, Over Expression, Activation Assay

    Activation of cAMP/PKA pathway induces ONH astrocytes death via Bax and caspase-3 activation a Real-time RT-PCR analysis of NFκB- and FoxO-dependent gene expression in ONH astrocytes treated with forskolin (10 μM) for 1 h. Data were normalized by GAPDH expression. b Real-time RT-PCR analysis of Bim mRNA expression in ONH astrocytes treated with forskolin (10 μM) or forskolin (10 μM) plus H89 (10 μM) for 1 h. Note that PKA dependent-regulation of Bim mRNA expression. c and d Cell viability and cell death analysis using MTT assay ( c ) and LDH assay (d) in ONH astrocytes co-treated with forskolin (10 μM) and IGF-1 (100 nM) for 24 h. e Co-IP analysis of Bim and Bcl-xL interaction in ONH astrocytes treated with forskolin (10 μM), H89 (10 μM) or forskolin (10 μM) plus H89 (10 μM) for 1 h. f Immunoblot analysis of Bim and activated Bax in ONH astrocytes co-treated with forskolin (10 μM) and rolipram (2 μM) for 24 h. g Immunoblot analyses of pAKT, AKT, activated Bax and caspase-3 in ONH astrocyte treated with forskolin (10 μM), Rp-cAMP (50 μM), or H89 (10 μM) for 24 h. For each determination, the mRNA, ratio of pAKT/AKT protein and other protein expression in controls was normalized to a value of 100% or 1.0. Data are shown as the mean ± S.D. ( n = 3). * P

    Journal: Cell Death & Disease

    Article Title: Elevated intracellular cAMP exacerbates vulnerability to oxidative stress in optic nerve head astrocytes

    doi: 10.1038/s41419-017-0171-8

    Figure Lengend Snippet: Activation of cAMP/PKA pathway induces ONH astrocytes death via Bax and caspase-3 activation a Real-time RT-PCR analysis of NFκB- and FoxO-dependent gene expression in ONH astrocytes treated with forskolin (10 μM) for 1 h. Data were normalized by GAPDH expression. b Real-time RT-PCR analysis of Bim mRNA expression in ONH astrocytes treated with forskolin (10 μM) or forskolin (10 μM) plus H89 (10 μM) for 1 h. Note that PKA dependent-regulation of Bim mRNA expression. c and d Cell viability and cell death analysis using MTT assay ( c ) and LDH assay (d) in ONH astrocytes co-treated with forskolin (10 μM) and IGF-1 (100 nM) for 24 h. e Co-IP analysis of Bim and Bcl-xL interaction in ONH astrocytes treated with forskolin (10 μM), H89 (10 μM) or forskolin (10 μM) plus H89 (10 μM) for 1 h. f Immunoblot analysis of Bim and activated Bax in ONH astrocytes co-treated with forskolin (10 μM) and rolipram (2 μM) for 24 h. g Immunoblot analyses of pAKT, AKT, activated Bax and caspase-3 in ONH astrocyte treated with forskolin (10 μM), Rp-cAMP (50 μM), or H89 (10 μM) for 24 h. For each determination, the mRNA, ratio of pAKT/AKT protein and other protein expression in controls was normalized to a value of 100% or 1.0. Data are shown as the mean ± S.D. ( n = 3). * P

    Article Snippet: The primary antibodies were mouse monoclonal anti-cAMP antibody (1:500; Sigma), mouse monoclonal anti-Bax antibody (6A7; 1:100; Santa Cruz), and rabbit polyclonal caspase-3 (1:100; Cell signaling).

    Techniques: Activation Assay, Quantitative RT-PCR, Expressing, MTT Assay, Lactate Dehydrogenase Assay, Co-Immunoprecipitation Assay

    Western blot analysis of protein levels of p53 and Bax. Actin, used as the internal control, was detected at the position corresponding to a molecular weight of 46 kDa. * Represents p

    Journal: Experimental Neurobiology

    Article Title: Protective Effect of Coriolus versicolor Cultivated in Citrus Extract Against Nitric Oxide-Induced Apoptosis in Human Neuroblastoma SK-N-MC Cells

    doi: 10.5607/en.2011.20.2.100

    Figure Lengend Snippet: Western blot analysis of protein levels of p53 and Bax. Actin, used as the internal control, was detected at the position corresponding to a molecular weight of 46 kDa. * Represents p

    Article Snippet: Mouse anti-p53 antibody (1 : 500; Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit anti-phospho p53 (Thr 18) antibody (1 : 200; Santa Cruz Biotech), and mouse anti-Bax antibody (1 : 1,000; Santa Cruz Biotech) were used as primary antibody.

    Techniques: Western Blot, Molecular Weight

    RT-PCR analysis of the mRNA levels of p53 and Bax. * Represents p

    Journal: Experimental Neurobiology

    Article Title: Protective Effect of Coriolus versicolor Cultivated in Citrus Extract Against Nitric Oxide-Induced Apoptosis in Human Neuroblastoma SK-N-MC Cells

    doi: 10.5607/en.2011.20.2.100

    Figure Lengend Snippet: RT-PCR analysis of the mRNA levels of p53 and Bax. * Represents p

    Article Snippet: Mouse anti-p53 antibody (1 : 500; Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit anti-phospho p53 (Thr 18) antibody (1 : 200; Santa Cruz Biotech), and mouse anti-Bax antibody (1 : 1,000; Santa Cruz Biotech) were used as primary antibody.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) increases the ratio of Bcl-2+/Bax+ cells after compression spinal cord injury (SCI). Rm-PgP (10 μg Rm) was injected immediately after clip compression injury. At 3 days post-injection, spinal cords were explanted, embedded, sectioned, and stained for Bax, pro-apototic protein and Bcl-2, anti-apoptotic protein. Sham and untreated SCI animal groups were used for comparison. Representative images of Bcl-2+ cells (A) and Bax+ cells (B) in the spinal cord at the lesion epicenter, 2, and 4 mm rostral and caudal. Scale bar indicates 50 μm, Top: sham. Middle: untreated SCI. Bottom: Rm-PgP treated SCI animals. (C) The ratio of Bcl-2+/Bax+ cells were calculated from a total of nine different sections of spinal cords from each group (three sections/rat, three rats/group). Sections were digitally imaged and the numbers of Bax+ and Bcl-2 + cells were counted. * p

    Journal: Journal of Neurotrauma

    Article Title: Rolipram-Loaded Polymeric Micelle Nanoparticle Reduces Secondary Injury after Rat Compression Spinal Cord Injury

    doi: 10.1089/neu.2017.5092

    Figure Lengend Snippet: Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) increases the ratio of Bcl-2+/Bax+ cells after compression spinal cord injury (SCI). Rm-PgP (10 μg Rm) was injected immediately after clip compression injury. At 3 days post-injection, spinal cords were explanted, embedded, sectioned, and stained for Bax, pro-apototic protein and Bcl-2, anti-apoptotic protein. Sham and untreated SCI animal groups were used for comparison. Representative images of Bcl-2+ cells (A) and Bax+ cells (B) in the spinal cord at the lesion epicenter, 2, and 4 mm rostral and caudal. Scale bar indicates 50 μm, Top: sham. Middle: untreated SCI. Bottom: Rm-PgP treated SCI animals. (C) The ratio of Bcl-2+/Bax+ cells were calculated from a total of nine different sections of spinal cords from each group (three sections/rat, three rats/group). Sections were digitally imaged and the numbers of Bax+ and Bcl-2 + cells were counted. * p

    Article Snippet: Mouse monoclonal anti-Bax and rabbit polyclonal anti-Bcl-2 primary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Injection, Cross-linking Immunoprecipitation, Staining

    Interaction of Bax with Bcl-2 in BL2 EBV (−) and BL2/B95 EBV (+) cells untreated or treated with nutlin-3. Cells were treated with 10 μ M nutlin-3 or the solvent dimethyl sulfoxide for 7 h. Lysates were subjected to immunoprecipitation with agarose-conjugated anti-Bax pAb (IP) or agarose-conjugated control rabbit IgG (IgG control) and then subjected to western blotting with an anti-Bcl-2 mAb or an anti-Bax pAb. As a control for protein levels before IP, a portion of cell lysate (input) corresponding to 15% of the input for IP was also included in the western blot. All results are representative of three independent experiments

    Journal: Cell Death & Disease

    Article Title: Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

    doi: 10.1038/cddis.2011.67

    Figure Lengend Snippet: Interaction of Bax with Bcl-2 in BL2 EBV (−) and BL2/B95 EBV (+) cells untreated or treated with nutlin-3. Cells were treated with 10 μ M nutlin-3 or the solvent dimethyl sulfoxide for 7 h. Lysates were subjected to immunoprecipitation with agarose-conjugated anti-Bax pAb (IP) or agarose-conjugated control rabbit IgG (IgG control) and then subjected to western blotting with an anti-Bcl-2 mAb or an anti-Bax pAb. As a control for protein levels before IP, a portion of cell lysate (input) corresponding to 15% of the input for IP was also included in the western blot. All results are representative of three independent experiments

    Article Snippet: Anti-Bax pAb (N-20), anti-Bcl-2 mAb (clone 100), anti-Mcl-1 mAb (clone 22), anti-Bcl-xl mAb (7B2.5) and anti-BZLF1 mAb (BZ1) were purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

    Techniques: Immunoprecipitation, Western Blot

    Effect of nutlin-3 treatment on Bax relocalization and activation in EBV (−) and EBV (+) cells. ( a ) Cells were treated with 10 μ M nutlin-3 or the solvent dimethyl sulfoxide for 24 h. Cytosolic and mitochondrial fractions were analyzed by western blotting with an anti-Bax pAb. Vinculin and Bcl-2 were used as cytosolic and mitochondrial markers, respectively. Fold-change values versus the untreated control (0 h), after normalization with respect to vinculin or Bcl-2 protein levels, are shown under the blots. Results are representative of three independent experiments. ( b ) BL2 EBV (−) cells and BL2/B95, LY47 and Remb1 EBV (+) cells were treated with 10 μ M nutlin-3 or the solvent dimethyl sulfoxide for 24 h. Cells were fixed in 0.25% paraformaldehyde and labeled with a conformation-specific 6A7 Bax antibody, which recognizes only the active conformation of the protein and Alexa 488-conjugated goat anti-mouse IgG. Cells were then analyzed with a FACSCalibur flow cytometer

    Journal: Cell Death & Disease

    Article Title: Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

    doi: 10.1038/cddis.2011.67

    Figure Lengend Snippet: Effect of nutlin-3 treatment on Bax relocalization and activation in EBV (−) and EBV (+) cells. ( a ) Cells were treated with 10 μ M nutlin-3 or the solvent dimethyl sulfoxide for 24 h. Cytosolic and mitochondrial fractions were analyzed by western blotting with an anti-Bax pAb. Vinculin and Bcl-2 were used as cytosolic and mitochondrial markers, respectively. Fold-change values versus the untreated control (0 h), after normalization with respect to vinculin or Bcl-2 protein levels, are shown under the blots. Results are representative of three independent experiments. ( b ) BL2 EBV (−) cells and BL2/B95, LY47 and Remb1 EBV (+) cells were treated with 10 μ M nutlin-3 or the solvent dimethyl sulfoxide for 24 h. Cells were fixed in 0.25% paraformaldehyde and labeled with a conformation-specific 6A7 Bax antibody, which recognizes only the active conformation of the protein and Alexa 488-conjugated goat anti-mouse IgG. Cells were then analyzed with a FACSCalibur flow cytometer

    Article Snippet: Anti-Bax pAb (N-20), anti-Bcl-2 mAb (clone 100), anti-Mcl-1 mAb (clone 22), anti-Bcl-xl mAb (7B2.5) and anti-BZLF1 mAb (BZ1) were purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

    Techniques: Activation Assay, Western Blot, Labeling, Flow Cytometry, Cytometry

    Effect of a combination of nutlin-3 and ABT-737 in the EBV (+) cell lines. BL2B95 EBV (+) cells were or were not subjected to prior treatment with 10 μ M ABT-737 for 1 h, and were then treated with nutlin-3 for 7 h. ( a ) The levels of Bcl-2 and Bax proteins were assessed by western blot analysis. ( b ) Lysates were subjected to immunoprecipitation with agarose-conjugated anti-Bax pAb (IP) or agarose-conjugated control rabbit IgG (IgG control) and then subjected to western blotting with an anti-Bcl-2 mAb or an anti-Bax pAb. As a control for protein levels before IP, a portion of cell lysate (Input) corresponding to 15% of the input for IP was also included in the western blot. All results are representative of three independent experiments. ( c ) BL2/B95 EBV (+) cells were or were not subjected to prior treatment with 10 μ M ABT-737 for 1 h and were then treated with nutlin-3 for the indicated periods of time. Cytosolic and mitochondrial fractions were analyzed by western blotting with an anti-Bax pAb. Vinculin and Bcl-2 were used as cytosolic and mitochondrial markers, respectively. Fold-change values versus the untreated control (0 h), after normalization with respect to the levels of vinculin or Bcl-2 protein, are shown under the blots. The results shown are representative of three independent experiments. ( d ) BL2/B95 cells were or were not subjected to prior treatment with ABT-737 for 1 h and were then left untreated or treated with nutlin-3 for 24 h. Cells were fixed in 0.25% paraformaldehyde and labeled with a conformation-specific 6A7 Bax antibody, which recognizes only the active conformation of the protein and Alexa 488-conjugated goat anti-mouse IgG. Cells were then analyzed with a FACSCalibur flow cytometer

    Journal: Cell Death & Disease

    Article Title: Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

    doi: 10.1038/cddis.2011.67

    Figure Lengend Snippet: Effect of a combination of nutlin-3 and ABT-737 in the EBV (+) cell lines. BL2B95 EBV (+) cells were or were not subjected to prior treatment with 10 μ M ABT-737 for 1 h, and were then treated with nutlin-3 for 7 h. ( a ) The levels of Bcl-2 and Bax proteins were assessed by western blot analysis. ( b ) Lysates were subjected to immunoprecipitation with agarose-conjugated anti-Bax pAb (IP) or agarose-conjugated control rabbit IgG (IgG control) and then subjected to western blotting with an anti-Bcl-2 mAb or an anti-Bax pAb. As a control for protein levels before IP, a portion of cell lysate (Input) corresponding to 15% of the input for IP was also included in the western blot. All results are representative of three independent experiments. ( c ) BL2/B95 EBV (+) cells were or were not subjected to prior treatment with 10 μ M ABT-737 for 1 h and were then treated with nutlin-3 for the indicated periods of time. Cytosolic and mitochondrial fractions were analyzed by western blotting with an anti-Bax pAb. Vinculin and Bcl-2 were used as cytosolic and mitochondrial markers, respectively. Fold-change values versus the untreated control (0 h), after normalization with respect to the levels of vinculin or Bcl-2 protein, are shown under the blots. The results shown are representative of three independent experiments. ( d ) BL2/B95 cells were or were not subjected to prior treatment with ABT-737 for 1 h and were then left untreated or treated with nutlin-3 for 24 h. Cells were fixed in 0.25% paraformaldehyde and labeled with a conformation-specific 6A7 Bax antibody, which recognizes only the active conformation of the protein and Alexa 488-conjugated goat anti-mouse IgG. Cells were then analyzed with a FACSCalibur flow cytometer

    Article Snippet: Anti-Bax pAb (N-20), anti-Bcl-2 mAb (clone 100), anti-Mcl-1 mAb (clone 22), anti-Bcl-xl mAb (7B2.5) and anti-BZLF1 mAb (BZ1) were purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

    Techniques: Western Blot, Immunoprecipitation, Labeling, Flow Cytometry, Cytometry

    SPL treatment prevents diabetes-induced glomerular and tubular damage. (A) Schematic representation of the experimental strategy used in this study. Four groups of rats were studied: Control (CTL), Diabetic (DBT), DBT+SPL and SPL, respectively (for detail see Material and methods ). (B) Purity of isolated glomerular and proximal tubular segments was evaluated by IB analysis of Wilm´s tumor 1 (WT1) and dipeptidylpeptidase (DppD) expressions, respectively. (C) IB analysis of nephrin and WT1 shows that SPL treatment prevents diabetes-induced glomerular damage; densitometric analysis of nephrin (D) and WT1 (E) are shown ( S1 Fig ). (F) IB analysis of kidney injury molecule 1 (Kim)-1 and heat shock protein of 72 KDa (Hsp72) show that SPL treatment decreases diabetes-induced proximal tubular damage, densitometric analysis of Kim-1 (G) and Hsp72 (H) are shown ( S1 Fig ). No changes were found in SPL group compared to CTL group. (I) IF analysis confirms that SPL treatment decreases diabetes-induced Kim-1 expression (green label) in PT labeled with DppD (red label). Nuclei were marked with 4', 6-diamidino- 2-phenylindole (DAPI, blue label). No significant changes were found between SPL and CTL groups. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control for IB. Data are mean±SEM from 3 rats per group. *p

    Journal: PLoS ONE

    Article Title: Aldosterone signaling regulates the over-expression of claudin-4 and -8 at the distal nephron from type 1 diabetic rats

    doi: 10.1371/journal.pone.0177362

    Figure Lengend Snippet: SPL treatment prevents diabetes-induced glomerular and tubular damage. (A) Schematic representation of the experimental strategy used in this study. Four groups of rats were studied: Control (CTL), Diabetic (DBT), DBT+SPL and SPL, respectively (for detail see Material and methods ). (B) Purity of isolated glomerular and proximal tubular segments was evaluated by IB analysis of Wilm´s tumor 1 (WT1) and dipeptidylpeptidase (DppD) expressions, respectively. (C) IB analysis of nephrin and WT1 shows that SPL treatment prevents diabetes-induced glomerular damage; densitometric analysis of nephrin (D) and WT1 (E) are shown ( S1 Fig ). (F) IB analysis of kidney injury molecule 1 (Kim)-1 and heat shock protein of 72 KDa (Hsp72) show that SPL treatment decreases diabetes-induced proximal tubular damage, densitometric analysis of Kim-1 (G) and Hsp72 (H) are shown ( S1 Fig ). No changes were found in SPL group compared to CTL group. (I) IF analysis confirms that SPL treatment decreases diabetes-induced Kim-1 expression (green label) in PT labeled with DppD (red label). Nuclei were marked with 4', 6-diamidino- 2-phenylindole (DAPI, blue label). No significant changes were found between SPL and CTL groups. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control for IB. Data are mean±SEM from 3 rats per group. *p

    Article Snippet: Mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), goat anti-cldn-8, goat anti-with-no-lysine kinase 4 (WNK4), goat anti-CD4, mouse anti-vascular endothelial (VE)-cadherin, rabbit anti-nephrin and anti-Wilm´s Tumor 1 (WT1) and peroxidase-conjugated anti-goat antibodies were purchased from Santa Cruz Biotech, Inc. (Santa Cruz, CA, USA).

    Techniques: CTL Assay, Isolation, Expressing, Labeling

    Bax translocates to mitochondria and forms high mol wt oligomers during anoikis. (a) FSK-7 cells, adherent or detached (maintained on poly-HEMA for 15 min or 4 h), were separated into soluble (S) and membrane (M) fractions before SDS-PAGE and immunoblotting with polyclonal anti-Bax (top). 4 mM CHAPS was added, and Bax was immunoprecipitated with anti-Bax 62M. Immunoprecipitates were separated by SDS-PAGE and were immunoblotted with anti-Bax 5B7 (bottom). (b) Cells expressing the OMM marker YFP-XT were detached for various times before cytospinning, and were immunostained with the activation-dependent antibody 62M. 62M reactivity is seen at discrete regions on mitochondria at all time points. Bar, 5 μm. (c) Soluble and membrane fractions from adherent cells or cells maintained on poly-HEMA for 4 h were isolated as above. CHAPS was added to a final concentration of 4 mM, and the fractions were concentrated. 5 mg protein from each fraction was loaded onto a Sephacryl S100-HR column (see Materials and methods). Separated fractions were analyzed by SDS-PAGE and immunoblotting for Bax. (d) Membrane fractions from FSK-7 cells detached for 4 h were separated by size-exclusion chromatography as above. Fractions containing high (oligo) and low (mono) mol wt Bax complexes were collected. Fractions were concentrated and separated into three equal parts. These were treated with BS3 (with or without addition of 0.1% Triton X-100). Samples were separated by SDS-PAGE and were immunoblotted for Bax. Cytosolic Bax from adherent cells (Adh) was run for comparison after identical treatment. (e) Membrane fractions isolated from adherent cells or cells detached for various times and separated by size-exclusion chromatography were analyzed by SDS-PAGE and immunoblotting for Bax. Total soluble (S) and membrane (M) fractions show Bax translocation at each time point. (f) Membrane fractions prepared from adherent cells or cells detached for 15 min, 1 h, or 4 h were spilt into three and treated with 5 mM BS3 in the presence or absence of 0.1% Triton-X100. Samples were analyzed by SDS-PAGE and were immunoblotted for Bax.

    Journal: The Journal of Cell Biology

    Article Title: Spatial and temporal changes in Bax subcellular localization during anoikis

    doi: 10.1083/jcb.200302154

    Figure Lengend Snippet: Bax translocates to mitochondria and forms high mol wt oligomers during anoikis. (a) FSK-7 cells, adherent or detached (maintained on poly-HEMA for 15 min or 4 h), were separated into soluble (S) and membrane (M) fractions before SDS-PAGE and immunoblotting with polyclonal anti-Bax (top). 4 mM CHAPS was added, and Bax was immunoprecipitated with anti-Bax 62M. Immunoprecipitates were separated by SDS-PAGE and were immunoblotted with anti-Bax 5B7 (bottom). (b) Cells expressing the OMM marker YFP-XT were detached for various times before cytospinning, and were immunostained with the activation-dependent antibody 62M. 62M reactivity is seen at discrete regions on mitochondria at all time points. Bar, 5 μm. (c) Soluble and membrane fractions from adherent cells or cells maintained on poly-HEMA for 4 h were isolated as above. CHAPS was added to a final concentration of 4 mM, and the fractions were concentrated. 5 mg protein from each fraction was loaded onto a Sephacryl S100-HR column (see Materials and methods). Separated fractions were analyzed by SDS-PAGE and immunoblotting for Bax. (d) Membrane fractions from FSK-7 cells detached for 4 h were separated by size-exclusion chromatography as above. Fractions containing high (oligo) and low (mono) mol wt Bax complexes were collected. Fractions were concentrated and separated into three equal parts. These were treated with BS3 (with or without addition of 0.1% Triton X-100). Samples were separated by SDS-PAGE and were immunoblotted for Bax. Cytosolic Bax from adherent cells (Adh) was run for comparison after identical treatment. (e) Membrane fractions isolated from adherent cells or cells detached for various times and separated by size-exclusion chromatography were analyzed by SDS-PAGE and immunoblotting for Bax. Total soluble (S) and membrane (M) fractions show Bax translocation at each time point. (f) Membrane fractions prepared from adherent cells or cells detached for 15 min, 1 h, or 4 h were spilt into three and treated with 5 mM BS3 in the presence or absence of 0.1% Triton-X100. Samples were analyzed by SDS-PAGE and were immunoblotted for Bax.

    Article Snippet: Polyclonal anti-Bax recognizing the whole molecule was purchased from Santa Cruz Biotechnology, Inc.

    Techniques: SDS Page, Immunoprecipitation, Expressing, Marker, Activation Assay, Isolation, Concentration Assay, Size-exclusion Chromatography, Translocation Assay

    Photomicrographies showing the experimental group of rat tongue carcinogenesis after 4 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for bax (c) immunohistochemistry for bcl-2; (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)

    Journal: Dental Research Journal

    Article Title: The influence of sleep deprivation on expression of apoptosis regulatory proteins p53, bcl-2 and bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

    doi:

    Figure Lengend Snippet: Photomicrographies showing the experimental group of rat tongue carcinogenesis after 4 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for bax (c) immunohistochemistry for bcl-2; (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)

    Article Snippet: The specimens were then incubated with either anti-Bcl-2 monoclonal antibody (Santa Cruz, Biotechnology, USA) at a concentration of 1:200, anti-Bax monoclonal antibody (Santa Cruz Biotechnology, USA)) at a concentration of 1:100 and anti-p53 antibody (Santa Cruz Biotechnology, USA) which is able to detect both wild-type and mutant isoforms, at a concentration of 1:100.

    Techniques: Staining, Immunohistochemistry, H&E Stain

    Photomicrographies showing the control group of rat tongue carcinogenesis after 12 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for bax (c) immunohistochemistry for bcl-2; (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)

    Journal: Dental Research Journal

    Article Title: The influence of sleep deprivation on expression of apoptosis regulatory proteins p53, bcl-2 and bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

    doi:

    Figure Lengend Snippet: Photomicrographies showing the control group of rat tongue carcinogenesis after 12 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for bax (c) immunohistochemistry for bcl-2; (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)

    Article Snippet: The specimens were then incubated with either anti-Bcl-2 monoclonal antibody (Santa Cruz, Biotechnology, USA) at a concentration of 1:200, anti-Bax monoclonal antibody (Santa Cruz Biotechnology, USA)) at a concentration of 1:100 and anti-p53 antibody (Santa Cruz Biotechnology, USA) which is able to detect both wild-type and mutant isoforms, at a concentration of 1:100.

    Techniques: Staining, Immunohistochemistry, H&E Stain

    Photomicrographies showing the experimental group of rat tongue carcinogenesis after 12 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for bax (c) immunohistochemistry for bcl-2; (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)

    Journal: Dental Research Journal

    Article Title: The influence of sleep deprivation on expression of apoptosis regulatory proteins p53, bcl-2 and bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

    doi:

    Figure Lengend Snippet: Photomicrographies showing the experimental group of rat tongue carcinogenesis after 12 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for bax (c) immunohistochemistry for bcl-2; (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)

    Article Snippet: The specimens were then incubated with either anti-Bcl-2 monoclonal antibody (Santa Cruz, Biotechnology, USA) at a concentration of 1:200, anti-Bax monoclonal antibody (Santa Cruz Biotechnology, USA)) at a concentration of 1:100 and anti-p53 antibody (Santa Cruz Biotechnology, USA) which is able to detect both wild-type and mutant isoforms, at a concentration of 1:100.

    Techniques: Staining, Immunohistochemistry, H&E Stain

    Photomicrographies showing the control group of rat tongue carcinogenesis after 4 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for bax (c) immunohistochemistry for bcl-2; (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)

    Journal: Dental Research Journal

    Article Title: The influence of sleep deprivation on expression of apoptosis regulatory proteins p53, bcl-2 and bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

    doi:

    Figure Lengend Snippet: Photomicrographies showing the control group of rat tongue carcinogenesis after 4 weeks-treatment (a) hematoxylin-eosin stain; (b) immunohistochemistry for bax (c) immunohistochemistry for bcl-2; (d) immunohistochemistry for p53 (hematoxylin and eosin stain; 100× magnification)

    Article Snippet: The specimens were then incubated with either anti-Bcl-2 monoclonal antibody (Santa Cruz, Biotechnology, USA) at a concentration of 1:200, anti-Bax monoclonal antibody (Santa Cruz Biotechnology, USA)) at a concentration of 1:100 and anti-p53 antibody (Santa Cruz Biotechnology, USA) which is able to detect both wild-type and mutant isoforms, at a concentration of 1:100.

    Techniques: Staining, Immunohistochemistry, H&E Stain