Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 1. Western blot analysis of CFTR and STAT1 expression in un- treated (lane 1), PMA-treated (lane 2), TNF-treated (lane 3), and IFN- treated (lane 4) T84 (A) and RCMC (B). T84 cells were treated with PMA (10 ng/ml), human recombinant TNF (10 ng/ml), or IFN (10 ng/ml) for 24 h. RCMC were treated with PMA (10 ng/ml) or rat recombinant TNF (10 ng/ml) or IFN (10 ng/ml) for 24 h. Cell lysates were resolved on an 4 to 12% SDS-PAGE and blotted with anti-CFTR, anti-STAT1, or anti-actin antibody. Western blots shown represent three independent experiments. The effect of 24 h IFN treatment (80 ng/ml) on CFTR expression was characterized in T84 cells (C) and rat peritoneal mast cells (D), using confocal laser scanning microscopy (bar on the figure represents 10 m).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Western Blot, Expressing, Recombinant, SDS Page, Confocal Laser Scanning Microscopy

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 4. A, IFN-mediated up-regulation of mast cell CFTR protein requires activation of JAK2 and/or p38 and ERK. RCMC, LAD2, and T84 were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SB202190; 30 mg/ml), or ERK MAPK inhibitor (U0126; 30 mg/ml) for 24 h. Representative of three inde- pendent experiments. B, densitometry summary of data in Fig. 4. RCMC were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SP202190; 30 mg/ml), or ERK inhibitor (U0126; 30 mg/ml) for 24 h, and expression of CFTR (black columns) and STAT1 (gray columns) was analyzed by Western blot (n 3 independent experiments; error bars represent S.E.M.). As- terisks represent statistical significance as determined by Student’s t test (p 0.05).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Western blotting of endogenous PACAP in CHO-K1 and neuro2a cells. As shown CHO-K1 had no detectable endogenous PACAP, while neuro2a cells produced endogenous PACAP. (B) The expression of PAC1-YFP and M-PAC1-YFP detected by immunofluorescence. Shown were immunofluorescence results of PAC1-CHO and M-PAC1-CHO cells cultured in DMEM with 0.5% CS-FBS at 37°C overnight, which indicated that both PAC1 and M-PAC1 trafficked normally to the plasma membrane and 0.5% CS-FBS induced no significant receptors endocytosis. (C) Fluorescence densities assays. Shown were the YFP fluorescence densities in the whole cell lysate detected using the Victor3 1420 multi-label counter with excitation (460±30 nm) and emission (535±30 nm), indicating that the expression levels of PAC1-YFP in CHO cells were equal to those of M-PAC1-YFP. (D) Western blotting assays using reductive SDS-PAGE. Western blotting with a goat polyclonal IgG against the C-terminus of PAC1 in the reductive condition showed that there were similar bands with the molecular weight about 160 kD in PAC1-CHO and M-PAC1-CHO, but not in CHO. (E) The cell viabilities of PAC1-CHO and M-PAC1-CHO cells promoted by PACAP. The data were plotted as the fold changes of the treatment without PACAP (0 nM). After the cells were submitted the addition of PACAP (1–100 nM) in the absence of CS-FBS for 24 h, MTT assays showed that PACAP exerted more significant proliferative effects on M-PAC1-CHO than on PAC1-CHO (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO), indicating that the activation level of PAC1 by PACAP was lower than that of M-PAC1. (F) The intracellular cAMP levels induced by PACAP (1–100 nM) in PAC1-CHO and M-PAC1-CHO cells. After the data were plotted as the fold changes of the treatment with 0 nM PACAP, it was shown that the intracellular cAMP levels in M-PAC1-CHO cells induced by PACAP were significantly higher than the intracellular cAMP levels in PAC1-CHO cells induced by PACAP (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Western Blot, Produced, Expressing, Immunofluorescence, Cell Culture, Clinical Proteomics, Membrane, Fluorescence, SDS Page, Molecular Weight, Activation Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) The remaining cell viabilities of PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells 48 h after serum withdrawal. When the data were plotted as the percentage of the initial cell viability without serum withdrawal, it was shown that PAC1-CHO had remaining cell viability (57.34±5.91%) that was significantly higher than that of M-PAC1-CHO (36.96±6.85%) or pcDNA-CHO (37.89±7.11%) (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). (B) The intracellular caspase3 activities after serum withdrawal. The reactions of pcDNA-CHO were considered not result from PAC1 because pcDNA-CHO did not express PAC1 or PACAP; therefore, all the data were plotted as fold changes in pcDNA-CHO. As shown, PAC1-CHO had significantly lower caspase3 activity than M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO), whereas there was no significant difference between M-PAC1-CHO and pcDNA-CHO. (C) The intracellular Bcl-2 levels after serum withdrawal. After the data were plotted as the fold changes of pcDNA-CHO, it was shown that PAC1-CHO had significantly higher Bcl-2 level about 2 folds of that in M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments. (D) The detection of β-catenin, cyclin D1 and c-myc levels in PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells by western blotting. The western blotting results and the statistical analysis showed that the levels of β-catenin, cyclin D1 and c-myc (tow targets of β-catenin) in PAC1-CHO cells were significantly higher than those in M-PAC1-CHO or pcDNA-CHO cells (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). These findings indicated that overexpression of wild type PAC1 endowed CHO with anti-apoptotic activities against serum withdrawal, suggesting that PAC1 had ligand independent basal activity, while M-PAC1 did not. And Wnt/β-catenin signals were involved in the anti-apoptotic activity of PAC1-CHO. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Activity Assay, Western Blot, Over Expression

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Knockdown of endogenous PACAP and PAC1 with shRNA in Neuro2a. Western blotting assays showed that shRNA against PACAP significantly diminished the expression of endogenous PACAP in neuro2a/PACAP - , and further transfection with shRNA plasmids against PAC1 (+) to neuro2a/PACAP - cells decreased the PAC1 levels significantly, while control plasmids (-) did not interfere with expression of PAC1. The knockdown of PACAP and PAC1 in neuro2a produced a chance for the detection of the correlation of PAC1 down-regulation with its ligand independent basal activity. (B) The remaining cell viabilities of nero2a/PACAP - transfected with PAC1 shRNA plasmids (+) or control plasmid (-). After the data were plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that down-regulation of PAC1 with PAC1 shRNA plasmids (+) decreased the remaining cell viabilities to almost a half of the remaining cell viabilities transfected with control plasmids (-) 48 h after serum withdrawal (*, P<0.01, shRNA + vs. shRNA-). (C) Western blotting of β-catenin, cyclin D1 and c-myc in the nero2a/PACAP - cells transfected with PAC1 shRNA plasmids (+) or control plasmids (-). After the relative protein levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that PAC1 shRNA plasmids (+) significantly decreased the levels of β-catenin, cyclin D1 and c-myc compared with control plasmids (+)(*, P<0.01, shRNA+ vs. shRNA-). These data suggested that down-regulation of PAC1 in the natural cells such neuro2a with high expression of PAC1 inhibited the anti-apoptotic activities in the ligand free condition. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Knockdown, shRNA, Western Blot, Expressing, Transfection, Control, Produced, Activity Assay, Plasmid Preparation

Journal: Advanced Science

Article Title: An Off‐the‐Shelf Artificial Proregenerative Macrophage for Pressure Ulcer Treatment

doi: 10.1002/advs.202415886

Figure Lengend Snippet: Preparation and characterization of engineered artM. A) Representative SEM images of PLGA and PLGA‐Lys, and TEM image of artM. B) Representative CLSM images of artM (blue: DiI; green: FITC; red: Cy5; M: cell membrane; L: lysate; P: PLGA). C) SDS‐PAGE of crucial membrane proteins (M: cell membrane; P: PLGA). D) Protein expression of CD11b, CD18, CD44 and CD14 determined by WB. E) The XPS spectrum of artM. F) The stability of artM in PBS (10% FBS) under storage for 5 days. G, H) IL‐10 (G) and vascular endothelial growth factor (VEGF) (H) analyzed by ELISA ( n = 4). W: week.

Article Snippet: To observe the structure and composition of artM, 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI, C1991S, Beyotime, Shanghai, China), Fluorescein isothiocyanate (FITC, MCE) and Sulfo‐Cyanine5 (Cy5, MCE) were used to label the cell membrane, lysate and PLGA, respectively, and photographed with CLSM.

Techniques: Membrane, SDS Page, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: RNAi-mediated depletion of Rab7 causes Vps26 dissociation from endosomes. (A) HeLa cells were treated twice at 24-h intervals with a control, inactive siRNA, or siRNAs to Rab7, Rab4 (targeting both the a and b isoforms), or Vps26. Cell extracts were analyzed by SDS-PAGE and immunoblotting with antibodies to Rab7, Rab4, Vps26, or actin (loading control) as indicated in the figure. (B–Q) HeLa cells treated as in A with control (B, C, F, G, J, K, N, and O) or Rab7 siRNA (D, E, H, I, L, M, P, and Q) were immunostained for Vps26 (B, D, F, H, J, L, N, and P) and AP-3 (C and E), AP-1 (G and I), SNX1 (K and M), and SNX2 (O and Q) using rabbit polyclonal antibody to Vps26 and mouse monoclonal antibodies to AP-3–δ, AP-1–γ1, SNX1, or SNX2 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG and Alexa Fluor 488–conjugated donkey anti–mouse IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, SDS Page, Western Blot, Bioprocessing, Epifluorescence Microscopy

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Specificity and rescue of Vps26 dissociation from membranes in RNAi-treated cells. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A) or siRNAs to both Rab4a and Rab4b (B) or to Rab7 (C–F). At 36 h after the second treatment with siRNA to Rab7, some cells were transfected with plasmids encoding RNAi-resistant (i.e., canine) GFP-Rab7 (C and E) or GFP (D and F). Examples of cells expressing GFP or canine GFP-Rab7 are indicated by arrows. The cellular distribution of Vps26 (A–D) was assessed by indirect immunofluorescent staining using rabbit polyclonal antibody to Vps26 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Transfection, Expressing, Staining, Epifluorescence Microscopy

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Depletion of Rab7 blocks the transport of CI-MPR from endosomes to the TGN. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A, C, E, and G) or siRNA to Rab7 (B, D, F, and H–N). At 48 h after treatment, the steady-state distribution of CI-MPR (A–D) was assessed by indirect immunofluorescent staining of fixed cells using rabbit polyclonal antibody to cytosolic tail of CI-MPR followed by Alexa Fluor 488–conjugated donkey anti–rabbit IgG. C and D correspond to high magnification views of CI-MPR–positive structures from control or Rab7-depleted cells, respectively. Live control cells (E and G) or Rab7-depleted cells (F and H–N) were incubated with an antibody to the luminal domain of CI-MPR for 2 h at 37°C. Cells were washed, fixed, permeabilized, and stained with Alexa Fluor 488–conjugated donkey anti–mouse IgG to detect internalized antibody to CI-MPR (E–H, J, and M). G and H show high magnification views from control (E) or Rab7-depleted cells (F), respectively. Cells in I–N were additionally stained with rabbit polyclonal antibody to TfR (I–K) or giantin (L–N) followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images in A–H were captured using an epifluorescence microscope, and images in I–N were captured with a confocal microscope. (K and N) For merged images, yellow indicates colocalization. Arrows in I–K indicate examples of foci where proteins colocalize. (O) Extracts of HeLa cells treated with siRNAs to the proteins indicated on top were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting (IB) with antibodies to the proteins indicated on the right. Equal amounts of total protein were loaded. Bars: (A, B, E, and F) 15 μm; (C, D, G, and H) 1.5 μm; (I–N) 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Staining, Incubation, Microscopy, SDS Page, Western Blot

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Rab7 depletion impairs processing of cathepsin D. HeLa cells were treated twice at 24-h intervals with inactive siRNA (control; lane 1) or siRNAs to the proteins indicated on top. At 24 h after the second round of siRNA treatment, cells were rinsed with PBS and incubated in serum-free culture medium for 24 h. The medium was collected and precipitated with trichloroacetic acid, and the resulting pellets were dissolved in Laemmli sample buffer. Cell extracts and media samples were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting with rabbit polyclonal antibody to cathepsin D. Equal amounts of total protein were loaded on each lane. Blots were also probed with antibody to actin as a loading control. The positions of molecular mass markers (in kilodaltons) and of the precursor (pCatD), intermediate (iCatD), and mature (mCatD) forms of cathepsin D are indicated.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Incubation, SDS Page, Western Blot

Journal: Clinical Cancer Research

Article Title: NF-Y–Dependent Cyclin B2 Expression in Colorectal Adenocarcinoma

doi: 10.1158/1078-0432.ccr-06-1461

Figure Lengend Snippet: Fig. 1. Tissue expression of cyclin B2. A, human adult tissue poly(A)+ Northern blots were probed with 32P-labeled cyclin B2 (CCNB2) cDNA. B, differential expression of cyclin B2 in colorectal adenocarcinomas and nontumor tissues.The extracted proteins (40 Ag)were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Bottom, the blot was incubated with the primary cyclin B2 antibody and then with the actin antibody as a loading control. C, protein lysates were prepared from 293Tcells transfected with the Myc vector alone (lane1) or Myc-tagged cyclin B2 (lane 2).The membranes were incubated with either anti-Myc or anti ^ cyclin B2 antibodies. Arrow, cyclin B2 protein. Numbers on the left, molecular weight markers.

Article Snippet: Human colorectal cancer cells (HT29, NCI-H630, NCI-H508, SNU-C2A, and NCIH498) were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Expressing, Northern Blot, Labeling, Quantitative Proteomics, SDS Page, Membrane, Incubation, Control, Transfection, Plasmid Preparation, Molecular Weight

Journal: Clinical Cancer Research

Article Title: NF-Y–Dependent Cyclin B2 Expression in Colorectal Adenocarcinoma

doi: 10.1158/1078-0432.ccr-06-1461

Figure Lengend Snippet: Fig. 3. Immunohistochemical detection of cyclin B2 in the normal colorectal mucosa and colorectal adenocarcinoma. A, no cyclin B2^ positive cells were detected in the normal colorectal epithelium. Original magnification, 40. B, cyclin B2 staining in a well-differentiated adenocarcinoma was localized mainly in the cytoplasm of the epithelial cells.The immunoreactivity had a stippled or spotted pattern and was barely detectable in the adjacent lamina propria, muscularis mucosa, submucosal vessels, or fibrous tissue. Original magnification, 100. C, cyclin B2^ positive cells throughout the moderately differentiated adenocarcinoma epithelium. Original magnification, 100. D, staining for cyclin B2 was detected in the poorly differentiated adenocarcinoma associated with considerable stromal fibrosis of the colorectum. Original magnification, 200. E, staining for cyclin B2 was detected in the mucinous adenocarcinoma but not in the mucin pool and surrounding stromal cells. Original magnification, 200. F, Kaplan-Meier plot of the overall survival of patients with colorectal adenocarcinoma grouped based on cyclin B2 expression.The difference between groups was significant (P = 0.050, Mantel-Cox log-rank test).

Article Snippet: Human colorectal cancer cells (HT29, NCI-H630, NCI-H508, SNU-C2A, and NCIH498) were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Clinical Cancer Research

Article Title: NF-Y–Dependent Cyclin B2 Expression in Colorectal Adenocarcinoma

doi: 10.1158/1078-0432.ccr-06-1461

Figure Lengend Snippet: Fig. 2. Cytoplasmic expression of the cyclin B2 protein. A, the cells (HT29) were transfected with1 Ag of the GFP-fused cyclin B2 expression vector or an empty vector pEGFP-C2.The cells were processed for1 Ag/mL Hoechst 33258 staining to visualize the nuclei (blue) and for indirect immunofluorescence staining of cyclin B2 (red).The cells were stained with 1 Ag/mL Hoechst 33258 to visualize the nuclei (blue).The cell morphology was observed using laser scanning microscopy (LCM510).Trans, transmission. Bar, 10 Am. B, endogenous cyclin B2 stained with anti ^ cyclin B2 (red).The HT29 cells were stained with1 Ag/mL Hoechst 33258 to visualize the nuclei (blue). Bar, 10 Am. C, indirect immunofluorescence microscopy of human colorectal adenocarcinoma cells (NCI-H630). Endogenous cyclin B2 expression at various stages of mitosis. P, prophase; M, metaphase; A, anaphase; T, telophase. Bar, 10 Am.

Article Snippet: Human colorectal cancer cells (HT29, NCI-H630, NCI-H508, SNU-C2A, and NCIH498) were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Immunofluorescence, Laser-Scanning Microscopy, Transmission Assay, Microscopy

Journal: Clinical Cancer Research

Article Title: NF-Y–Dependent Cyclin B2 Expression in Colorectal Adenocarcinoma

doi: 10.1158/1078-0432.ccr-06-1461

Figure Lengend Snippet: Fig. 4. Transcriptional regulation of cyclin B2 by NF-Y. A, inhibition of cyclin B2 transcription by a NF-YA dominant-negative vector.The colorectal carcinoma cells were transfected with the promoters from cyclin B2 driving luciferase transcription and 3 Ag of the mutant-type NF-YA expression plasmids (YA13m29).The assay was done 24 h after transfection. Columns, mean of quadruplicate cultures from three independent experiments; bars, SE. *, P < 0.05. B, cell growth of the four types of colorectal cells. At the indicated time intervals, the monolayer cells were processed for the 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide assay and the absorbance was read at a wavelength of 540 nm using an ELISA reader. Points, mean of quadruplicate cultures from two independent experiments; bars, SE. *, P < 0.05. C, chromatin immunoprecipitation assays showing binding of NF-Y subunits to the cyclin B2 promoter. M, marker. Lane1, NF-YA subunit; lane 2, NF-YA + NF-YB + NF-YC subunits; lane 3, control; lane 4, input. D, a mutation of the CCAAT boxes impairs the cyclin B2 promoter activity.The B2-Luci vector (500 ng) carrying the wild-type murine cyclin B2 promoter, theY1,2m vector carrying a mutation in two CCAAT boxes, or theY1,2,3m vector carrying a mutation in all three CCAAT boxes was transiently cotransfected in the four types of colorectal cells. Columns, mean of quadruplicate cultures from three independent experiments; bars, SE.

Article Snippet: Human colorectal cancer cells (HT29, NCI-H630, NCI-H508, SNU-C2A, and NCIH498) were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Inhibition, Dominant Negative Mutation, Plasmid Preparation, Transfection, Luciferase, Mutagenesis, Expressing, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Binding Assay, Marker, Control, Activity Assay

Journal: Clinical Cancer Research

Article Title: NF-Y–Dependent Cyclin B2 Expression in Colorectal Adenocarcinoma

doi: 10.1158/1078-0432.ccr-06-1461

Figure Lengend Snippet: Fig. 5. Immunofluorescence assay of the NF-Y subunits in colorectal adenocarcinoma cells. A, NCI-H508 cells were transfected with1 Ag of the expression vector of the GFP-fused NF-YA, red fluorescent protein (RFP)^ fused NF-YB, GFP-fused NF-YC, or an empty vector pEGFP-C2.The cells were processed for indirect immunofluorescence (IF) staining for NF-YA (red), NF-YB (green), and NF-YC (red).The cells were stained with 1 Ag/mL Hoechst 33258 to visualize the nuclei (blue). Bar, 10 Am. B, endogenous NF-Y subunits stained with anti-NF-YA (red), anti-NF-YB (green), and anti-NF-YC (red). NCI-H508 cells were stained with 1 Ag/mL Hoechst 33258 to visualize the nuclei (blue). Bar,10 Am. C, the NCI-H508 cells were cotransfected with red fluorescent protein ^ fused NF-YB expression vector and GFP-fused NF-YC expression vector together. Bar,10 Am.

Article Snippet: Human colorectal cancer cells (HT29, NCI-H630, NCI-H508, SNU-C2A, and NCIH498) were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Staining

Journal: Clinical Cancer Research

Article Title: NF-Y–Dependent Cyclin B2 Expression in Colorectal Adenocarcinoma

doi: 10.1158/1078-0432.ccr-06-1461

Figure Lengend Snippet: Fig. 6. Immunohistochemical reactivity of NF-Y subunits in colorectal adenocarcinoma. A and E, cytoplasmic immunoreactivity for cyclin B2 was positive in the adenocarcinoma epithelium but negative in the normal epithelium at the crypt base. B and F, positive nuclear and cytoplasmic immunoreactivity for NF-YA can be seen in the adenocarcinoma epithelium as well as in the normal epithelium at the crypt base. C and G, positive nuclear and cytoplasmic immunoreactivity for NF-YA can be seen in the adenocarcinoma epithelium as well as in the normal epithelium at the crypt base. D and H, cytoplasmic immunoreactivity for NF-YC was positive in the adenocarcinoma epithelium but negative in the normal epithelium at the crypt base.

Article Snippet: Human colorectal cancer cells (HT29, NCI-H630, NCI-H508, SNU-C2A, and NCIH498) were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Immunohistochemical staining

Journal: Applied Microbiology and Biotechnology

Article Title: New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii

doi: 10.1007/s00253-016-7354-6

Figure Lengend Snippet: Analysis of hGH expression under the control of the different promoter/5′UTR elements. a PCR confirmation of gene integration and homoplasmy of hGH transformants. Three-primer PCR demonstrating the homoplasmic integration of hGH expression cassettes into the C. reinhardtii chloroplast genome. The TN72 cell line was transformed with plasmids that introduced the hGH into the plastome downstream of psbH as illustrated in Fig. . PCR of genomic DNA using primers F1, R1, and R3 ( hGH.R ) give a 0.88-kb product for the TN72 plastome, and a 1.5–2.0-kb product for the transgenic plastomes depending on the size of the promoter/5′UTR element. The absence of the 0.88-kb band indicates that the transformant lines are homoplasmic. M = DNA ladder of size markers. b Western blot analysis of the C. reinhardtii transformants. Crude protein extracts, equalized according to the optical density of the cultures, were fractionated by SDS-PAGE and probed with antibodies to hGH. Control = a TN72 transformant generated using the empty pSRSapI vector. Pure recombinant hGH was run alongside to aid quantification of the 22-kDa protein. An identical gel was blotted and probed with antibodies to the large subunit ( RbcL ) of the endogenous Rubisco enzyme as a loading control. Relative band intensities of hGH were quantified using an Odyssey Infrared Imaging System and scaled to give 1.000 for the atpA element. c Western blot analysis of the E. coli transformants. Control = an E. coli transformant generated using the empty pSRSapI vector

Article Snippet: The probed blots were scanned and quantified using an Odyssey Infrared Imaging System (LI-COR).

Techniques: Expressing, Transformation Assay, Transgenic Assay, Western Blot, SDS Page, Generated, Plasmid Preparation, Recombinant, Imaging

Journal: PLoS ONE

Article Title: Methionine Sulfoxide Reductase A (MsrA) Deficient Mycoplasma genitalium Shows Decreased Interactions with Host Cells

doi: 10.1371/journal.pone.0036247

Figure Lengend Snippet: A. Immunoblot analysis. Cytosol and membrane fractions were prepared from M. genitalium strains, separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-MsrA ( I ) and anti-EF-G ( II ). G37 and MS5 represent M. genitalium wild type and msrA mutant strains, respectively. W, M and C indicate whole, membrane and cytosol fractions of M. genitalium . MsrA indicates the bands reacting with anti-MsrA antiserum. EF-G indicates the bands reacting with anti-EFG antiserum. B. Immunohistochemical localization of MsrA. M. genitalium (G37 and MS5) cells were suspended uniformly, fixed with paraformaldehyde and permeabilized with Triton X-100 and Tween-20 or not permeabilized. This was followed by incubation in anti-MsrA antiserum and fluorescein conjugated anti-rabbit IgG secondary antibody. Images were captured with a FV 1000 confocal laser scanning microscope using 60× objective with 5× electronic zoom. FLUOR and DIC represent fluorescent and differential interference contrast images of the same field. G37, M. genitalium wild type strain; MS5, M. genitalium msrA mutant strain.

Article Snippet: The bacterial cells were washed three times with PBS followed by staining with Fluorescein anti-rabbit IgG (Vector, Burlingame, CA) for 2 h in room temperature.

Techniques: Western Blot, SDS Page, Mutagenesis, Immunohistochemical staining, Incubation, Laser-Scanning Microscopy

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Cx43 expression and GJ function of HCT116 and CT26 cells, including parental, control-shRNA, and Cx43-shRNA transfected cells. RES, resveratrol-treated cells. All other groups were compared with the Parental group using one-way ANOVA. * P < 0.05 represents a significant difference from values in the Parental group. SEMs are shown as error bars. (A1) Western blot images of Cx43 and phospho-Cx43 (Ser-368). β-Actin is used as a loading control. The full-length blots are shown as . (A2,A3) Bar diagrams of densitometric analysis. Protein expression level is normalized by β-actin. (B) GJ function analysis by “Parachute” dye-coupling assay. Scale bars are 20 μm. Columns show the mean ± SEM. All images of “Parachute” dye-coupling assay are shown as .

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: Expressing, Control, shRNA, Transfection, Western Blot

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Results of the MTT assay and CalcuSyn software analysis for parental, shRNA transfected, and Cx43 transfected HCT116 and CT26 cells. vector, pTARGET vector; CTX, cetuximab treatment; RES, resveratrol treatment; High, high density cultured cells (80%); Low, low density cultured cells (5%). (A) MTT assay results. The vertical axis represents growth inhibition rate, which is compared with that of untreated cells. The horizontal axis represents agent concentrations. Points represent mean ± SEM. Panels (A13–A16) are the results of high density culture cells. The raw data of MTT are shown as . (B) CalcuSyn software analysis of the MTT assay results. CI, combination index. CI is calculated by calcusyn analysis based on the inhibition rate of different concentration of resveratrol and cetuximab. CI > 1 represents antagonistic cytotoxicity; CI = 1 represents addictive cytotoxicity; CI < 1 represents synergistic cytotoxicity. The data generated by CalcuSyn are shown as . (C) IC50 (μg/ml) of cells to resveratrol and/or cetuximab. * P < 0.05 represents a significant difference between parental and transfected cells compared by one-way ANOVA. There is no significant difference between parental and control-shRNA in all conditions. The statistical difference of growth inhibition between parental and Cx43-shRNA transfected cells in different concentration is shown as .

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: MTT Assay, Software, shRNA, Transfection, Plasmid Preparation, Cell Culture, Inhibition, Concentration Assay, Generated, Control

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Immunoprecipitation analysis of Akt and Cx43 in parental HCT116 and CT26 cells. Cell lysates were subjected to immunoprecipitation with anti-Akt or anti-Cx43 antibodies. Then immunoprecipitated proteins were examined by western blotting with anti-Cx43 or anti-Akt antibodies, respectively. The input of cell total protein was used as a positive control and rabbit IgG was used as a negative control. Molecular weight of Akt and IgG heavy chain is similar, so they are hardly to be discriminated. SDS-PAGE of immunoprecipitation analysis is shown as . (A) IB: Cx43; IP: Akt. (A1) Non-treated cells. (A2) Resveratrol-treated cells. (A3) Cetuximab-treated cells. (A4) Cetuximab + Resveratrol-treated cells. (B) IB: Akt; IP: Cx43. (B1) Non-treated cells. (B2) Resveratrol-treated cells. (B3) Cetuximab-treated cells. (B4) Cetuximab + Resveratrol-treated cells.

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: Immunoprecipitation, Western Blot, Positive Control, Negative Control, Molecular Weight, SDS Page

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Western blot analysis of proteins related to the EGFR signal pathway, including EGFR, p-EGFR, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, IKKα, IκBα, NFκB p65, MAPK, and p-MAPK. (A) Western blot images. β-Actin is used as a loading control for total protein, and histone H3 is used as a loading control for nuclear proteins. CTX, cetuximab treatment; RES, resveratrol treatment. The full-length blots are shown as . (B) Relationship between the EGFR signaling pathway and Cx43 ( , , ). Cetuximab inhibits the binding reaction of EGF to EGFR, which inhibits the whole EGFR signaling pathway. Akt, a key molecule in this pathway, interacts with Cx43. Resveratrol increases Cx43 expression and phosphorylation.

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: Western Blot, Control, Binding Assay, Expressing, Phospho-proteomics

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Inhibition of tumor growth by cetuximab and resveratrol. CTX, cetuximab-treated mice; RES, resveratrol-treated mice. (A) The subcutaneous tumor model was created by parental, control-shRNA, or Cx43-shRNA-transfected cells. Each group contains five Balb/c mice for CT26 or nu/nu nude mice for HCT116. These mice were then treated with cetuximab and/or resveratrol for 14 days before sacrificed. Tumors were removed, and weight was measured using electronic balance. (B) Columns represent mean ± SEM volume from five tumors. Groups “CTX,” “RES,” and “CTX+RES” were compared with group “No treated” using one-way ANOVA. Blue * P < 0.05 represents a significant difference. Group “CTX+RES” was compared with group “CTX” and “RES” using one-way ANOVA. Yellow * P < 0.05 represents a significant difference.

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: Inhibition, Control, shRNA, Transfection

Journal: Oncogene

Article Title: DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

doi: 10.1038/s41388-018-0404-z

Figure Lengend Snippet: Polγ nitration and activity. a Detection of Polγ nitration after UVB irradiation (50 mJ/cm 2 × 1 h) in human primary epidermal keratinocytes using 3-nitrotyrosine immunoprecipitation followed by western blotting with Polγ antibody or Polγ antibody-mediated immunoprecipitation followed by western blotting with 3-nitotyrosine antibody. Both IgG and inputs were provided as loading control. b Detection of Polγ nitration by immunoprecipitation after UVB treatment (50 mJ/cm 2 ) in JB6 cells using 3-nitrotyrosine antibody with exposure to authentic peroxynitrite (ONOO − ) as a positive control. Both IgG and inputs were provided as loading control. c Polγ activity in human keratinocytes with or without UVB treatment was detected by 20% acrylamide/7 M urea gel electrophoresis and autoradiography following primer extension using mtDNA-specific primers (see Materials and methods). d Polγ activity in JB6 cells was detected as described in panel ( c ). e SDS-PAGE of purified recombinant mouse Polγ protein and visualization by Coomassie brilliant blue staining. f Purified Polγ proteins were immunoprecipitated with 3-nitrotyrosine antibody before and after treatment with peroxynitrite (250 μM). Polγ proteins were detected by western blotting using an anti-Polγ antibody. Both IgG and inputs were used as loading control. g Activity of purified Polγ protein was measured by dTTP incorporation in the presence or absence of peroxynitrite as described in the Materials and methods. h Nitration of tyrosine residues was detected by mass spectrometry. Each peak in the MS/MS spectrum represents relative abundance of peptide fragment and the tyrosine residues of the peptide fragment are shown under each graph. For all quantifications ( c , d ), each data point represents the mean ± SD of three individual samples. For all panels, representative figures from three repeated experiments are shown. Statistical analysis was performed using t tests. Statistical significance is indicated by asterisks: * p < 0.05 and ** p < 0.01

Article Snippet: Polγ siRNA (Cat. Sc-155884) and the lentiviral Polγ shRNA plasmid (Sc-155884-SH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Nitration, Activity Assay, Irradiation, Immunoprecipitation, Western Blot, Control, Positive Control, Nucleic Acid Electrophoresis, Autoradiography, SDS Page, Purification, Recombinant, Staining, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: Oncogene

Article Title: DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

doi: 10.1038/s41388-018-0404-z

Figure Lengend Snippet: Effects of Polγ suppression on mitochondria. a The mouse Polγ gene structure in chromosome 7 is shown schematically. The vertical bars indicate the exons. The exon 3 targeted by CRISPR is highlighted in red. Two 20-nucleotide guide sequences of CRISPR are shown, in blue and in green. The pam sequences are underlined, and red triangles indicate the incision sites by Cas9n. b Polγ protein levels were measured by western blotting in PCRISPR JB6 clones. c Polγ activity in mitochondria isolated from PCRISPR cells was detected by primer extension followed by autoradiography (see Materials and methods). d The PCR product ratio of long-amplicon (LA) mtDNA to short-amplicon (SA) mtDNA in control and Polγ-deficient PCRISPR cells which is normalized by mitochondrial mass. e The number of DNA lesions per 10 kb mtDNA in Polγ-deficient PCRISPR cells following normalization with mitochondrial mass. Note that the mitochondrial masses of control and PCRISPR cells were obtained by staining the cells with NAO, a mitochondria-specific dye, followed by flow cytometry. f Mitochondrial oxygen consumption was measured as described in Materials and Methods. OCR oxygen consumption rate. g Glycolysis was measured as described in Materials and Methods. ECAR extracellular acidification rate. h The level of mitochondrial reactive oxygen was measured by quantifying the MitoSox Red fluorescence in Polγ-deficient PCRISPR cells in the presence and absence of MnP (MnTnBuOE-2-PyP 5+ ), with positive and negative controls. The mean fluorescence intensity of MitoSox Red was determined using flow cytometry. The concentration of cellular superoxide was estimated by quantification of fluorescence intensity. Rotenone was used as a positive control for generation of ROS. PEG-SOD (Superoxide dismutase–polyethylene glycol from bovine erythrocytes) was also used as a control to remove superoxide generated by MitoSox Red. The fluorescence intensity of MitoSox Red is normalized by total mitochondrial mass. In all bar graphs and line graphs, each data point represents the mean ± SD of three individual samples. Each experiment was repeated at least three times and statistical analysis was performed using t tests for two groups or one-way ANOVA analysis and Bonferroni’s post-test for multiple-group comparisons. Statistical significance is indicated by asterisks: * p < 0.05 and ** p < 0.01

Article Snippet: Polγ siRNA (Cat. Sc-155884) and the lentiviral Polγ shRNA plasmid (Sc-155884-SH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: CRISPR, Western Blot, Clone Assay, Activity Assay, Isolation, Autoradiography, Amplification, Control, Staining, Flow Cytometry, Fluorescence, Concentration Assay, Positive Control, Generated

Journal: Oncogene

Article Title: DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

doi: 10.1038/s41388-018-0404-z

Figure Lengend Snippet: Suppression of Polγ increases the autophagic response. a LC3 punctation was detected in Polγ-deficient PCRISPR cells following expression of GFP-LC3. For each cell type, 100 GFP-positive cells were counted, and the right panel shows the quantification of punctated cells. b Western blot analysis and quantification of the increase in LC3 II, beclin 1, phosphorylated beclin 1, ATG7, and the decrease in p62 in Polγ-deficient PCRISPR cells compared to controls. c Autophagy flux was detected by western blotting in PCRISPR cells following treatment with autophagy inhibitors (MnP; MnTnBuOE-2-PyP 5+ 3-MA; 3-methyle adenine and Bafilomycin). The bar graph shows the quantification of LC3 II band intensity normalized to β-actin. d LC3 punctation was detected using fluorescence microscopy with or without autophagy inhibitors. The bar graph shows the quantification of punctated cells (100 GFP-positive cells were counted for each cell type). In all panels, each experiment was repeated at least three times. In the bar graphs, each data point represents the mean ± SD of three individual samples. Statistical analysis was performed using t tests for two groups or one-way ANOVA analysis and Bonferroni’s post-test for multiple-group comparisons. Statistical significance is indicated by asterisks: * p < 0.05 and ** p < 0.01

Article Snippet: Polγ siRNA (Cat. Sc-155884) and the lentiviral Polγ shRNA plasmid (Sc-155884-SH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Fluorescence, Microscopy

Journal: Oncogene

Article Title: DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

doi: 10.1038/s41388-018-0404-z

Figure Lengend Snippet: Effects of tyrosine mutation on Polγ activity and autophagy. a , panel a The activity of Polγ in mitochondria following transfection of wild-type and mutant (Y964F) Polγ expression vectors in Polγ-deficient PCRISPR cells. Relative activity of Polγ was measured and expressed as the fold change (bottom panel). The specificity of Polγ activity was validated by comparing with negative control, which contains template−primer mixture and radio-labeled ATP without enzyme. Panel b The ectopic expression of wild-type and mutant Polγ was detected by western blotting using Flag antibody. Endogenous level of Polγ in PCRISPR cells is also shown. b , panel a The Polγ activity in JB6 cells following coexpression of wild-type or mutant (Y964F) Polγ plasmid along with Polγ siRNA. Relative activity of Polγ was measured and expressed as the fold change (bottom panel). Panel b The suppression of Polγ by siRNA and the overexpression of wild-type and mutant Polγ were confirmed by western blotting using Polγ antibody. The relative level of LC3 II formation in JB6 cells following coexpression of wild-type or mutant (Y964F) Polγ plasmid along with Polγ siRNA or control siRNA is shown (bottom panel). The specificity of Polγ activity was validated by comparing with negative control, which contains template−primer mixture and radio-labeled ATP without enzyme. Each data point represents the mean ± SD of three individual samples. Statistical analysis was performed using one-way ANOVA analysis and Bonferroni’s post-test for multiple-group comparisons. Statistical significance is indicated by asterisks: * p < 0.05 and ** p < 0.01

Article Snippet: Polγ siRNA (Cat. Sc-155884) and the lentiviral Polγ shRNA plasmid (Sc-155884-SH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Mutagenesis, Activity Assay, Transfection, Expressing, Negative Control, Labeling, Western Blot, Plasmid Preparation, Over Expression, Control

Journal: Oncogene

Article Title: DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

doi: 10.1038/s41388-018-0404-z

Figure Lengend Snippet: Activation of AKT/mTOR pathway following Polγ deficiency. a , b Increased levels of mTOR phosphorylation at S2481, AKT phosphorylation at S473, and Rictor were detected by western blotting in a Polγ-deficient PCRISPR cells and b cells stably expressing Polγ shRNA. c , d In vitro mTOR kinase activities were measured by using specific substrates for mTOR complexes coupled with immunoprecipitation and western blotting in cells stably expressing Polγ shRNA. c For mTORC2 kinase activity, total cell lysates were immunoprecipitated with Rictor antibody. Panel a The immunoprecipitated proteins were detected by western blotting using Rictor antibody. Panel b Immunoprecipitated product was then incubated with purified AKT. The phosphorylated AKT (S473) was detected as an indicator of mTORC2 kinase activity. d , Panel a The immunoprecipitated proteins were detected by western blotting using Raptor antibody. For mTORC1 kinase activity, total cell lysates were immunoprecipitated with raptor antibody. Immunoprecipitated product was then incubated with purified p70S6K protein and the phosphorylated p70S6K was detected by western blotting as an indicator of mTORC1 activity. e Coimmunoprecipitation of Rictor and Raptor with mTOR from the total cell lysates of cells expressing Polγ shRNA. Input controls are provided as loading control. f Reverse coimmunoprecipitation with Rictor antibody followed by western blotting performed with Rictor, mTOR, and Raptor antibodies. Input controls were provided as loading control. g Immunoprecipitation was also carried out using mTOR antibody in JB6 cells stably expressing Polγ shRNA following pretreatment with MnP (20 μM × 24 h). The immunoprecipitated products were analyzed by western blotting using antibodies against mTOR and Rictor. Input controls were added as loading control. Similarly, reverse immunoprecipitation was performed using Rictor antibody in Polγ-deficient cells following pretreatment with MnP. Input controls are also shown as loading control. All immunoprecipitated proteins were normalized with house-keeping loading control and quantified. Each experiment was repeated at least three times. In the bar graphs, each data point represents the mean ± SD of three individual samples. Statistical analysis was performed using t tests for two groups or one-way ANOVA analysis and Bonferroni’s post-test for multiple-group comparisons. Statistical significance is indicated by asterisks: * p < 0.05 and ** p < 0.01

Article Snippet: Polγ siRNA (Cat. Sc-155884) and the lentiviral Polγ shRNA plasmid (Sc-155884-SH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Stable Transfection, Expressing, shRNA, In Vitro, Immunoprecipitation, Activity Assay, Incubation, Purification, Control

Journal: Oncogene

Article Title: DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

doi: 10.1038/s41388-018-0404-z

Figure Lengend Snippet: Polγ deficiency-mediated autophagy is RICTOR-dependent. a Western blotting shows the overexpression of Rictor or Raptor following transfection of the corresponding vectors in JB6 cells. AKT phosphorylation at Ser473 in the presence of the overexpressed Rictor was also detected by western blotting. Plasmid vector without any protein expression sequence was used as control and is designated by EV. b Detection of LC3 punctation following overexpression of Rictor or Raptor. Quantification of LC3 punctation in Raptor- and Rictor-overexpressing JB6 cells is shown (bottom). c Quantification of LC3 punctation in wild-type MEF and Rictor-knockout MEF cells following Polγ siRNA transfection. d Effects of Polγ deficiency in MEF and Rictor-knockout cells are achieved by expression of Polγ siRNA. The endogenous autophagy markers (LC3 II, beclin 1, ATG7, and p62) were detected by western blotting. The right panels show the corresponding quantification. e Overexpression of Rictor or Raptor in Rictor-knockout MEF cells and the level of LC3 II were determined by western blotting. AKT phosphorylation at Ser473 induced by Rictor overexpression was also detected by western blotting. In all panels, each experiment was repeated at least three times, and statistical analysis was performed using one-way ANOVA analysis followed by Bonferroni’s post-test for multiple-group comparisons. Statistical significance is indicated by asterisks: * p < 0.05 and ** p < 0.01

Article Snippet: Polγ siRNA (Cat. Sc-155884) and the lentiviral Polγ shRNA plasmid (Sc-155884-SH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Over Expression, Transfection, Phospho-proteomics, Plasmid Preparation, Expressing, Sequencing, Control, Knock-Out

Journal: Oncogene

Article Title: DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

doi: 10.1038/s41388-018-0404-z

Figure Lengend Snippet: Deficiency of Polγ enhances autophagy-dependent cell proliferation. a The cytotoxicity of ROS and autophagy inhibitors (MnP, 20 μM; 3-MA, 2.5 mM; and Bafilomycin, 50 nM) was assessed in JB6 cells 24 h after treatment by determining cell growth. b Cell growth was also measured in PCRISPR cells following treatment with the indicated inhibitor. c BrdU uptake was determined as a measure of cell proliferation in PCRISPR cells with or without ROS or autophagy inhibitors. d Phosphorylation of p70S6K was detected by western blotting in wild-type and Rictor-knockout MEF cells following suppression of Polγ by siRNA. e Cell proliferation was determined in Rictor-knockout cells after Polγ suppression using the BrdU uptake assay (assay details are given in Materials and Methods section). f Cell proliferation was also determined in Polγ-knockdown cells following treatment with AKT inhibitor using the BrdU uptake assay. In all panels, each data point represents the mean ± SD of three individual samples. Statistical analysis was performed using one-way ANOVA analysis and Bonferroni’s post-test for multiple-group comparisons. Statistical significance is indicated by asterisks: * p < 0.05 and ** p < 0.01

Article Snippet: Polγ siRNA (Cat. Sc-155884) and the lentiviral Polγ shRNA plasmid (Sc-155884-SH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Western Blot, Knock-Out, Knockdown

Journal: Oncogene

Article Title: DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

doi: 10.1038/s41388-018-0404-z

Figure Lengend Snippet: Summary of the proposed role of Polγ in prosurvival autophagy. Schematic diagram of the impact of Polγ deficiency and subsequent loss of enzymatic activity on the mTORC2/AKT pathway during prosurvival autophagy

Article Snippet: Polγ siRNA (Cat. Sc-155884) and the lentiviral Polγ shRNA plasmid (Sc-155884-SH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay