Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 3. Involvement of EGFR/AKT/CREB pathway in REG4-mediated macrophage polarization to M2. (A) Representative western blots of phosphorylated EGFR, AKT and CREB in macrophage cultures treated with REG4 (10 nM) for different time intervals indicated. (B) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively. Data are mean ± SEM from four independent experiments. *p<0.05, **P<0.01 compared to untreated cells. (C) Representative western blotting images showing the phosphorylation of EGFR, AKT and CREB in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. (D) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively, in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. Data are mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with REG4 treatment alone. (E) Representative western blots of CD163, CD68, and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. (F) Quantitative analysis of the immunoblots of CD163, CD68 and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. Data are mean ± SEM from three independent donors. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Phospho-proteomics, Infection, Control, shRNA

Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 4. The conditioned medium of Panc1 cells induces macrophage polarization to M2. (A) Western blots of REG4 in the culture medium or cell lysate of Panc1, AsPC1 and BxPC3 cells. Shown are representatives of three independent experiments with similar results. (B) Representative western blots of CD206 and CD163 in macrophage cultures treated with or without the conditioned medium of Panc1, AsPC1 and BxPC3 cell cultures, respectively. (C) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with or without the conditioned medium of Panc1 (CMPanc1), AsPC1 (CMAsPC1) and BxPC3 (CMBxPC3) cell cultures, respectively. Data are shown by mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with CMPanc1 treatment. (D) Western blots of REG4 in the culture medium and cell lysate of Panc1 cells infected with or without lentiviral REG4 shRNA. Shown are representatives of three independent experiments with similar results. (E) Representative western blots of CD206 and CD163 in macrophage cultures treated with the conditioned medium Panc1 cells infected with lentiviral control shRNA (CMPanc1/shCon) or REG4 shRNA (CMPanc1/shREG4). (F) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with CMPanc1/shCon or CMPanc1/shREG4. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Infection, shRNA, Control

Journal: BMC Genomics

Article Title: Comparative genomics of 84 Pectobacterium genomes reveals the variations related to a pathogenic lifestyle

doi: 10.1186/s12864-018-5269-6

Figure Lengend Snippet: Genes of T6SSc presence in the 61 Enterobacterales genomes. A total of 61 of the 203 Enterobacterales genomes possessed the T6SSc cluster. The strains with red letters were from the newly sequenced genomes in this study; the others strains were from NCBI. The 28 genes of T6SSc of the BCS7 strain was used as a standard; we grouped the genes into two Blocks (Block I includes the first 8 genes (starting from the 5′ end) of the cluster; Block II includes the 20 terminal genes of the cluster at the 3′ end). Map showing the presence (black feature) or absence (grey feature) of each gene of the T6SSc cluster, with a maximum likelihood phylogenetic tree based on the core genes of 61 Enterobacterales genomes. Colored arrows indicate five clusters that divide these strains into different known families ( Enterobacteriaceae , Erwiniaceae, Morganellaceae & Yersiniaceae , Budviciaceae & Hafniaceae , and Pectobacteriaceae ) of the Enterobacterales order. a and b: two different sequenced genomes (accession numbers: NZ_JIBO00000000 and NZ_FNQS00000000) of the Lonsdalea quercina subsp. quercina strain ATCC 29281 T , respectively. The frequency percentage of each gene of T6SSc among these genomes is shown on the right side next to the physical map

Article Snippet: Colored arrows indicate five clusters that divide these strains into different known families ( Enterobacteriaceae , Erwiniaceae, Morganellaceae & Yersiniaceae , Budviciaceae & Hafniaceae , and Pectobacteriaceae ) of the Enterobacterales order. a and b: two different sequenced genomes (accession numbers: NZ_JIBO00000000 and NZ_FNQS00000000) of the Lonsdalea quercina subsp. quercina strain ATCC 29281 T , respectively.

Techniques: Blocking Assay

Journal: Molecular Medicine Reports

Article Title: Angiopoietin-like protein 2 expression is suppressed by angiotensin II via the angiotensin II type 1 receptor in rat cardiomyocytes

doi: 10.3892/mmr.2016.5544

Figure Lengend Snippet: Effects of various angiotensin II (AngII) concentrations on cardiomyocytes. (A) mRNA expression levels of angiopoietin-like protein 2 (Angptl2) were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) following treatment with 100 nmol/l AngII for 48 h. cDNA sample size was 100 ng. Protein expression levels of Angptl2 were detected by western blotting (B) following treatment with 100 nmol/l AngII for various durations (0, 6, 24 and 48 h; protein sample size, 100 μ g), (C) or with various concentrations of AngII (0, 50, 100 or 200 nmol/l) for 24 h (protein sample size, 40 μ g). Enzyme-linked immunosorbent assay for Angptl2 was used to verify the trend detected by RT-qPCR and western blotting following treatment of cells with (D) various concentrations of AngII (E) for various durations (sample size of each group, 10 μ l). (F) Immunofluorescence was used to confirm the conclusions of these experiments. Green staining, Angptl2; blue staining, 4′,6-diamidino-2-phenylindole (DAPI); magnification, ×400. (G) Fluorescence intensity was analyzed using ImageJ software. * P<0.05, ** P<0.01 and *** P<0.001, vs. the corresponding control group (0 h or 0 nmol/l).

Article Snippet: The cells were then incubated with α-SMA (1:200; cat. no. 6487; Cell Signaling Technology, Inc., Danvers, MA, USA) and Angptl2 antibodies (1:400; cat. no. sc-292811; Santa Cruz Biotechnology, Inc.) overnight in a humidified incubator at 4°C.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence, Software, Control

Journal: Molecular Medicine Reports

Article Title: Angiopoietin-like protein 2 expression is suppressed by angiotensin II via the angiotensin II type 1 receptor in rat cardiomyocytes

doi: 10.3892/mmr.2016.5544

Figure Lengend Snippet: Role of angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) antagonists. To determine the possible underlying mechanisms associated with the effects of angiotensin II (AngII) on angiopoietin-like protein 2 (Angptl2), the cells were divided into six groups: Normal, AngII (100 nmol/l), AngII (100 nmol/l) + losartan (10 −4 mol/l), AngII (100 nmol/l) + PD123319 (10 −4 mol/l), normal + losartan (10 −4 mol/l) and normal + PD123319 (10 −4 mol/l). (A and B) Angptl2 protein expression was detected in each group by western blotting (protein sample size, 40 μ g). (C) Angptl2 mRNA expression was detected in each group by reverse transcription-quantitative polymerase chain reaction (cDNA sample size, 100 ng), (D) enzyme-linked immunosorbent assay (culture media volume, 10 μ l) and (E) immunofluorescence. (F) ImageJ software was used to analyze fluorescence intensity; blue and green fluorescence were assessed simultaneously (magnification, ×200). Data are presented as the mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001, comparison indicated by brackets. ns, not significant. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The cells were then incubated with α-SMA (1:200; cat. no. 6487; Cell Signaling Technology, Inc., Danvers, MA, USA) and Angptl2 antibodies (1:400; cat. no. sc-292811; Santa Cruz Biotechnology, Inc.) overnight in a humidified incubator at 4°C.

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Software, Fluorescence, Standard Deviation, Comparison

Journal: Oncology reports

Article Title: PKA/CREB regulates the constitutive promoter activity of the USP22 gene.

doi: 10.3892/or.2015.3740

Figure Lengend Snippet: Figure 3. CREB regulates the transcription of USP22 in HeLa cells. (A) Interference efficiencies of CREB were analyzed by western blot analysis with anti-CREB and anti-GAPDH antibodies. (B) HeLa cells were transiently transfected with siRNA against CREB (CREB siRNA) and negative control siRNA. USP22 promoter activity was measured by luciferase activity 24 h post-transfection. Data are representative of three independent experiments (P<0.01). (C) HeLa cells were transiently transfected with CREB siRNA and negative control siRNA. USP22 mRNA levels were measured via qRT-PCR 24 h post- transfection, and the results were normalized to GAPDH. (D) HeLa cells were co-transfected with CREB siRNA and the p-210/CREBmut plasmid, and USP22 promoter activity was measured by luciferase activity 24 h post-transfection. (E) CREB protein were examined via western blot analysis after pCDNA/CREB was transfected into the HeLa cells. (F) HeLa cells were co-transfected with CMV-driven CREB expression plasmids (pCDNA/CREB) and USP22 promoter constructs. Luciferase activities were measured 24 h post-transfection. (G) HeLa cells were transiently transfected with pcDNA3.1 empty vector or pcDNA/ CREB, with USP22 mRNA quantified via qRT-PCR 24 h post-transfection, and the results were normalized to GAPDH.

Article Snippet: One day before transfection, HeLa cells were plated in 24-well plates at a density of 5x104 cells/well, and then transfected with 20 nM of siControl Non-Targeting siRNA duplex (siControl) or human CREB1-specific siRNA duplex (sc-29281; Santa Cruz Biotechnology, Santa Cruz, CA, USA), using an siRNA reagent system (Santa Cruz Biotechnology) according to the manufacturer's instructions.

Techniques: Western Blot, Transfection, Negative Control, Activity Assay, Luciferase, Quantitative RT-PCR, Plasmid Preparation, Expressing, Construct

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 2. GIP modulates CREB/TORC2 activity in human adipocytes. Human adipocytes were serum starved in DMEM/Ham’s F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP or GLP-1 (100 nM) in the presence of insu- lin (1 nM). A: Effects of GIP on phosphorylation of CREB and nuclear localization of TORC2. Nuclear extracts were isolated and Western blot analyses performed with antibodies against phosphorylated Ser133-CREB, CREB, TORC2, and Histone H3. Cyto- plasmic extracts were immunoprecipitated (IP) with phospho-Serine/Threonine followed by immuno- blotting (IB) for TORC2. Input represents one-tenth of total cytoplasmic extracts used in assay. B: Confo- cal microscopy. Human adipocytes were treated as described above. Immunocytochemical staining was performed using antibodies against phospho-CREB (Ser133, green) or TORC2 (red) and nuclei were stained with DAPI (blue). The scale bar indicates 20 m and all imaging data were analyzed using the Northern Eclipse program (ver.6). Shown are repre- sentative of n = 3.

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Activity Assay, Phospho-proteomics, Isolation, Western Blot, Immunoprecipitation, Microscopy, Staining, Imaging, Northern Blot

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 3. CREB/TORC2 are functionally involved in the regulation of LPL expression. A: Effects of different CREB constructs on GIP-responsiveness of the LPL promoter. GIPR-HEK-293 cells were cotransfected with an LPL promoter reporter construct (chr8+: 19840052 - 19841249; -992 to +206 of LPL gene, 2 µg), and various CREB constructs or control vector pCMV5 (1 µg). After transfection, cells were treated with insulin (1 nM) plus GIP (100 nM), and the reporter activities are shown as the relative luciferase activity normalized to protein concentration. All data represent three independent experiments, each carried out in triplicate. Signifi cance was tested using ANOVA with Newman-Keuls post hoc test, where ** represents P < 0.05 versus untreated Vector Control, ## represents P < 0.05 versus respective untreated Control. B, C: RNAi-mediated suppression of CREB and TORC2 reduces GIP-stimulated LPL expression. B: Effects of CREB and TORC2 siRNAs on GIP-stimulated LPL protein expression. Human adipocytes were transfected with a pool of three siRNAs for CREB and TORC2 and incubated for 72 h. At 48 h after transfection, cells were treated with in- sulin (1 nM) plus GIP (100 nM) for 24 h and Western blot analyses were performed as described in Materials and Methods, using antibody against phospho-CREB (Serine-133), CREB, TORC2, LPL, Histone H3, and -actin. C. Effects of CREB and TORC2 siRNAs on GIP-stimulated LPL mRNA expression. Human adipo- cytes were treated as described above and real-time RT-PCR performed to quantify LPL mRNA levels; shown as the fold difference versus control normalized to 18S rRNA expression levels. Signifi cance was tested using ANOVA with Newman-Keul’s post hoc test where ** represents P < 0.05 versus Control siRNA, ## represents P < 0.05 versus GIP.

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Expressing, Construct, Control, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Protein Concentration, Incubation, Western Blot, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 4. GIP regulates CREB/TORC2 through a pathway involving PI3-K/PKB/AMPK. Human adipocytes were serum starved in DMEM/ Ham’s F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM). A: Effect of PI3-K inhibition on GIP-mediated decreases in phospho-AMPK Thr172 and nuclear CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of PI3-K inhibitors, LY 294002 (40 M) or wortman- nin (400 nM). B: Effect of AMPK modulation on CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of AMPK inhibitor (Compound C, 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]3-pyridin-4-yl- pyrrazolo[1,5-a]pyrimidine, 40 M) or AMPK activator (5-(3-(4-(2-(4-Fluorophenyl)ethoxy)phenyl)propyl)furan-2-carboxylic acid, FPPF, 50 M). AMPK inhibitor or activator was added to cells during 1 h preincubation as well as GIP stimulation. Nuclear/cytoplasmic extracts were isolated from each sample and Western blot analyses were performed. TORC2 and histone H3 blots are from nuclear extracts and phospho-AMPK Thr172 and AMPK blots are from cytoplasmic extracts. C: Effect of AMPK modulation on LPL activity. Human adipocytes were treated as described above and LPL activity was determined as described in Materials and Methods. D: Effect of GIP or GLP-1 on cAMP accumulation. Human adipocytes were treated as described above and incubated with GIP or GLP-1 (100 nM) in the presence of insulin

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Inhibition, Activity Assay, Isolation, Western Blot, Incubation

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 6. GIP increases protein-protein interaction between phospho-CREB and TORC2 in the nucleus. A: Coimmunoprecipitation. Human adipocytes were serum-starved in DMEM/Ham’s F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM) and in the presence or absence of PI3-K inhibitors, LY 294002 (40 M), wortmannin (400 nM) or AMPK activator (50 M). Nuclear extracts were isolated from each sample and immunoprecipitated (IP) with phospho-CREB (Ser133) followed by immunoblotting (IB) for TORC2. Input represents one-tenth of total nuclear extract used in the coimmunoprecipitation assay. B: Confocal microscopy. Human adipocytes were treated as described above and fi xed following stimulation with GIP (100 nM) in the presence or absence of PI3-K inhibitors or AMPK activator. Immunocytochemical staining was performed using antibodies against phospho-CREB (Ser133, green) or TORC2 (red) and nuclei were stained with DAPI (blue). The scale bar indicates 20 m and all imaging data were analyzed using the Northern Eclipse program (ver.6). Shown are representative of n = 3.

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Isolation, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Confocal Microscopy, Staining, Imaging, Northern Blot

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 7. Phospho-CREB/TORC2 binds CRE-II se- quence of human LPL promoter. A: Identifi cation of putative CRE sequences in the promoters of several adipocyte-specifi c genes. Potential CREs present in these promoters are indicated by the box-enclosed regions and consensus CRE sequences shown at the top of the fi gure. B, C: Representative gel of a ChIP assay for the binding of phospho-CREB (B) and TORC-2 (C) in the human LPL promoter. Human adipocytes were serum-starved in DMEM/Ham’s F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the pres- ence of insulin (1 nM). Phospho-CREB and TORC2 were respectively immunoprecipitated from intact chromatin isolated from human adipocytes using anti-phospho-CREB (Ser 133) and anti-TORC2 anti- body. Precipitated DNA fragments were analyzed by PCR using primers fl anking the CRE-I and CRE-II sites in the LPL promoter. An isotype-matched IgG was used as negative control and 1% Input (PCR product of one-hundredth of the total isolated DNA used in the ChIP assay) as positive control, respec- tively. Shown are representative of n = 3.

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Binding Assay, Immunoprecipitation, Isolation, Negative Control, Positive Control

Journal: Journal of Lipid Research

Article Title: GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

doi: 10.1194/jlr.m006841

Figure Lengend Snippet: Fig. 8. Proposed pathway by which GIP increases LPL activity in adipocytes. GIP receptor interaction results in PI3-K activation, increased phosphorylation of Ser473 and Thr308 in PKB, and reduced phosphorylation of Ser428 in LKB1 and Thr172 in AMPK. An associated de- crease in AMPK phosphorylation leads to dephosphorylation of TORC2, thus allowing increased translocation from the cytoplasm and into the nucleus. PKB also increases phospho-CREB Ser133 levels in the nucleus. TORC2 complexes with phospho-CREB Ser133 in the nucleus and binds to CRE-II of the human LPL promoter, thus turning on the transcriptional machinery for up-regulation of LPL .

Article Snippet: RNA interference knockdown of CREB and TORC2 To determine the effect of reducing levels of endogenous CREB and TORC2 on LPL expression, resuspended human adipocytes were transfected with a pool of 3 siRNAs each for CREB or TORC2 (Santa Cruz, sc-29281 and sc-45832) that resulted in 36 to 43% reduction of the respective mRNAs when applied as individual pools (data not shown).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, De-Phosphorylation Assay, Translocation Assay