double stranded dna oligonucleotide  (Macrogen)

Journal: International Journal of Molecular Sciences

Article Title: Dioxin-Induced PAI-1 Expression: A Novel Pathway to Pancreatic β-Cell Failure in Type 2 Diabetes

doi: 10.3390/ijms252211974

Figure Lengend Snippet: TCDD induced expression and secretion of PAI-1 in Hepa1c1c7 cells. ( A , B ) Dose-dependent changes of mRNA. Hepa1c1c7 cells were incubated with TCDD 0, 10, 50, 100, or 1000 pM for 24 h, and mRNA levels of CYP1A1 ( A ) and PAI-1 ( B ) were analyzed by qRT-PCR. ( C , D ) Time-dependent changes of mRNA. Hepa1c1c7 cells were incubated with 100 pM TCDD for 0, 4, 6, 24, or 48 h, and mRNA levels of CYP1A1 ( C ) and PAI-1 ( D ) were analyzed by qRT-PCR. The mRNA level was normalized by 18S rRNA. Data represent the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO-treated cells. ( E ) Hepa1c1c7 cells were incubated with TCDD for 24 h as designated, and the cell lysates were analyzed by Western blot using an antibody against PAI-1. β-Actin was used as a loading control. ( F ) Cells were incubated with DMSO or TCDD (100 or 1000 pM) in MEMα media containing 10% CS-FBS for 24 h. The culture media were changed to serum-free RPMI 1640, and the cells were incubated for 24 h. Proteins in the collected conditioned media were precipitated by MeOH/CHCl 3 and analyzed by Western blot using anti-PAI-1 antibody. The Ponceau S-stained image was used as a loading control.

Article Snippet: For AhR knock-down in Hepa1c1c7 cells, double-stranded DNA oligonucleotides for short hairpin RNA against mouse AhR (shAhR) containing the BamHI-shAhR (sense)-XhoI loop-antisense with T5-EcoRI sequences (5′-gatccg actctctgttcttaggctc ttctcgaga gagcctaagaacagagagt tttttggaag-3′) were cloned into the RNAi-Ready pSIREN-RetroQ vector (Clontech, Mountain View, CA, USA).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Dioxin-Induced PAI-1 Expression: A Novel Pathway to Pancreatic β-Cell Failure in Type 2 Diabetes

doi: 10.3390/ijms252211974

Figure Lengend Snippet: AhR-dependent induction of PAI-1 by TCDD. ( A , B ) Hepa1c1c7 cells were transfected with shSCR or shAhR. The expression of AhR was analyzed by Western blot ( A ) and intensities of AhR bands normalized by β-Actin ( B ). ( C , D ) Dose-dependent changes of mRNA. shSCR- or shAhR-transfected Hepa1c1c7 cells were incubated with TCDD as indicated for 24 h, and mRNA levels of CYP1A1 ( C ) and PAI-1 ( D ) were analyzed by qRT-PCR. ( E , F ) Time-dependent changes of mRNA. shSCR- or shAhR-transfected Hepa1c1c7 cells were incubated with 100 pM TCDD for 0, 4, 6, 24, or 48 h, and mRNA levels of CYP1A1 ( E ) and PAI-1 ( F ) were analyzed by qRT-PCR. The mRNA levels were normalized by 18S rRNA. Data represent the mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO-treated cells. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. shSCR. ( G ) shSCR- or shAhR-transfected Hepa1c1c7 cells were incubated with TCDD at the indicated concentrations for 24 h and analyzed by Western blot using anti-PAI-1 antibody. β-Actin was used as a loading control. ( H ) shSCR- or shAhR-transfected Hepa1c1c7 cells were incubated with DMSO or TCDD (100 or 1000 pM) in MEMα containing 10% CS-FBS for 24 h, followed by further incubation in serum-free RPMI 1640 for 24 h to collect conditioned media. Proteins in the conditioned media were precipitated by MeOH/CHCl 3 and analyzed by Western blot using anti-PAI-1 antibody. The Ponceau S-stained image was used as a loading control.

Article Snippet: For AhR knock-down in Hepa1c1c7 cells, double-stranded DNA oligonucleotides for short hairpin RNA against mouse AhR (shAhR) containing the BamHI-shAhR (sense)-XhoI loop-antisense with T5-EcoRI sequences (5′-gatccg actctctgttcttaggctc ttctcgaga gagcctaagaacagagagt tttttggaag-3′) were cloned into the RNAi-Ready pSIREN-RetroQ vector (Clontech, Mountain View, CA, USA).

Techniques: Transfection, Expressing, Western Blot, Incubation, Quantitative RT-PCR, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Dioxin-Induced PAI-1 Expression: A Novel Pathway to Pancreatic β-Cell Failure in Type 2 Diabetes

doi: 10.3390/ijms252211974

Figure Lengend Snippet: PAI-1 secreted by TCDD from Hepa1c1c7 cells induced apoptosis in RIN-m5F cells. ( A ) Graphical scheme of the conditioned media transfer experiment. Hepa1c1c7 cells were incubated with DMSO or 100 pM TCDD in MEMα containing 10% CS-FBS for 24 h. Conditioned media were collected 24 h after media change to serum-free RPMI 1640, and RIN-m5F cells were incubated in the conditioned media containing 10% CS-FBS for 24 h, with or without PAI-1 antibody. ( B ) After incubation with the conditioned media from shSCR- or shAhR-transfected Hepa1c1c7 cells, the RIN-m5F cells were stained with Annexin V and propidium iodide (PI), and apoptosis was monitored through flow cytometry. The X and Y axes show Annexin V and PI staining markers, for apoptosis and necrosis, respectively. Each plot is divided into four regions. The lower-left region shows cells negative for both Annexin V and PI (living cells); the lower-right region shows cells positive for Annexin V only (early apoptotic cells); the upper-left region shows cells positive for PI only (necrotic cells); the upper-right region shows cells positive for both. ( C ) Quantitative graph of early apoptotic cells as % of total cells. ( D ) Western blot against Caspase-3 and Caspase-8 after incubation with conditioned media for 24 h (C: control for incubation with conditioned media). β-Actin was used as a loading control. ( E , F ) After incubation in conditioned media, the cell viability and mitochondrial activity were measured by calcein ( E ) and intracellular ATP contents ( F ), respectively. ( G , H ) A Caspase-3/7 assay was performed after incubation with conditioned media from shSCR- or shAhR-transfected cells ( G ) or from Hepa1c1c7 cells with or without PAI-1 antibody ( H ). Data represent the mean ± SEM ( n = 3~5). * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. shSCR.

Article Snippet: For AhR knock-down in Hepa1c1c7 cells, double-stranded DNA oligonucleotides for short hairpin RNA against mouse AhR (shAhR) containing the BamHI-shAhR (sense)-XhoI loop-antisense with T5-EcoRI sequences (5′-gatccg actctctgttcttaggctc ttctcgaga gagcctaagaacagagagt tttttggaag-3′) were cloned into the RNAi-Ready pSIREN-RetroQ vector (Clontech, Mountain View, CA, USA).

Techniques: Incubation, Transfection, Staining, Flow Cytometry, Western Blot, Control, Activity Assay