sirna tdg  (Santa Cruz Biotechnology)

Journal: Epigenomes

Article Title: Modulation of DNA Methylation/Demethylation Reactions Induced by Nutraceuticals and Pollutants of Exposome Can Promote a C > T Mutation in the Breast Cancer Predisposing Gene PALB2

doi: 10.3390/epigenomes6040032

Figure Lengend Snippet: Diuron and PFOA supplementation affect APOBEC expression and TDG activity, respectively. ( A ) Impact of Diuron on the APOBEC3α and APOBEC3γ expression (ELISA method). Graph illustrates the fold change expression of APOBEC3α and APOBEC3γ expression seen in MCF10A cells exposed to Diuron at different doses (MAC and multiple of MAC). ”Ctrl” represents the control exposure performed with PBS/DMSO 5%. siRNA-A (control) and siRNA-APOBECs are used as control conditions inducing down-expression of APOBEC3α and APOBEC3γ. A significant difference (*) was observed between the different doses of Diuron used. ( B ) Impact of PFOA on TDG activity (Activity assay method). Graph illustrates the fold change of TDG activity seen in MCF10A cells exposed to PFOA at different doses (MAC and multiple of MAC). ”Ctrl” represents the control exposure performed with PBS/DMSO5%. A significant difference (*) was observed between the different doses of PFOA used.

Article Snippet: siRNA-APOBEC3γ (sc-60091, Santa Cruz, Heidelberg, Germany), siRNA-APOBEC3α (sc-72514, Santa Cruz, Heidelberg, Germany), siRNA-TDG (sc-44142, Santa Cruz, France), and siRNA-TET3 (sc-154206, Santa Cruz, Heidelberg, Germany) were transfected in MCF10A as previously described (Duforestel et al. 2019). siRNA-A (sc-37007, Santa Cruz, Heidelberg, Germany), that is, a scrambled sequence devoid of specific degradation of any cellular message was used as control.

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Epigenomes

Article Title: Modulation of DNA Methylation/Demethylation Reactions Induced by Nutraceuticals and Pollutants of Exposome Can Promote a C > T Mutation in the Breast Cancer Predisposing Gene PALB2

doi: 10.3390/epigenomes6040032

Figure Lengend Snippet: Ascorbic acid and Iron have the ability to limit the presence of the c.1027C > T mutation in PALB2 gene. ( A ) Impact of Glyphosate on TET3 expression (ELISA method). Graph illustrates the fold change expression of TET3 expression seen in MCF10A cells exposed to Glyphosate at different doses (MAC and multiple of MAC). “Ctrl” represents the control exposure performed with PBS/DMSO 5%. siRNA-A (control) and siRNA-TET3 are used as control conditions inducing down-expression of TET3. A significant difference (*) was observed between the different doses of glyphosate used. ( B ) Impact of Ascorbic Acid or Iron on TET activity (Activity assay method). Graph illustrates the fold change of TDG activity seen in MCF10A cells exposed to Ascorbic Acid or Iron at different doses (RDI and multiple of RDI). “Ctrl” represents the control exposure performed with PBS/DMSO 5%. DMOG was used as control condition inducing a decrease in TET activity since DMOG acts as a TET inhibitor. A significant difference (*) was observed between the different doses of ascorbic acid and iron used. ( C ) Graphs illustrate the Impact of nutraceuticals and pollutants as single agent or in a mixture on the presence of the c.1027C > T mutation in PALB2 gene (Restriction site mutation (RSM) method). Doses used are indicated in multiples of RDI or MAC. A significant difference (*) was observed between the indicated comparisons.

Article Snippet: siRNA-APOBEC3γ (sc-60091, Santa Cruz, Heidelberg, Germany), siRNA-APOBEC3α (sc-72514, Santa Cruz, Heidelberg, Germany), siRNA-TDG (sc-44142, Santa Cruz, France), and siRNA-TET3 (sc-154206, Santa Cruz, Heidelberg, Germany) were transfected in MCF10A as previously described (Duforestel et al. 2019). siRNA-A (sc-37007, Santa Cruz, Heidelberg, Germany), that is, a scrambled sequence devoid of specific degradation of any cellular message was used as control.

Techniques: Mutagenesis, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Viruses

Article Title: Porcine Circovirus Type 2 Rep Enhances IL-10 Production in Macrophages via Activation of p38-MAPK Pathway

doi: 10.3390/v11121141

Figure Lengend Snippet: Rep protein interacts with thymine DNA glycosylase (TDG) to enhance the binding activities of Sp1 and NF-κB p50 with the il10 promoter at the later phase of PCV2 infection. ( A ) These specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q were transfected to cells for 48 h, and the efficiency of each gene silencing was detected by western blotting. ( B ) The specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q siRNAs (siRNA #1 of c-Myc, siRNA #2 of ZNF265, siRNA #3 of TDG, and siRNA #2 of VG5Q) were transfected to PAMs, then rAd-Blank or rAd-Rep2 infected the cells (1 × 10 6 cells) for 48 h. The secretion of IL-10 was measured by ELISA in different siRNA-transfected-PAMs. ( C ) The secretion of IL-10 was detected in the TDG siRNA-transfected-PAMs in 0–24 h, 24–48 h, and 48–72 h after PCV2 inoculation by ELISA. The columns indicate IL-10 production in each 24 h in the culture supernatants. ( D ) The levels of IL-10 mRNA were detected in TDG siRNA-transfected-PAMs by Q-PCR with the same incubation time points as C. ( E , F ) TDG siRNA-transfected-PAMs were incubated with 5 MOI PCV2, the binding activities of NF-κB p50 and Sp1 to the il10 promoter were detected at 48 h by the ChIP assay. The data of ( A ) are representative of three independent experiments. The data of ( B – D ) are means ± SEM of three independent experiments. The data of ( E , F ) are the means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 versus NC siRNA-transfected cells in the same infection ( B , E , F ) or at the same time point ( C , D ).

Article Snippet: Cells were seeded overnight before transfection, allowed to reach 50% confluency by the time of transfection, and transfected with Akt siRNA, p38 siRNA, ERK siRNA, p50 siRNA, VG5Q siRNA, TDG siRNA, ZNF265 siRNA, and c-Myc siRNA ( ) (Sangon Biotech, Shanghai, China) using lipofectamine 2000 (Invitrogen), respectively.

Techniques: Binding Assay, Infection, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Viruses

Article Title: Porcine Circovirus Type 2 Rep Enhances IL-10 Production in Macrophages via Activation of p38-MAPK Pathway

doi: 10.3390/v11121141

Figure Lengend Snippet: Rep protein interacts with thymine DNA glycosylase (TDG) to enhance the binding activities of Sp1 and NF-κB p50 with the il10 promoter at the later phase of PCV2 infection. ( A ) These specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q were transfected to cells for 48 h, and the efficiency of each gene silencing was detected by western blotting. ( B ) The specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q siRNAs (siRNA #1 of c-Myc, siRNA #2 of ZNF265, siRNA #3 of TDG, and siRNA #2 of VG5Q) were transfected to PAMs, then rAd-Blank or rAd-Rep2 infected the cells (1 × 10 6 cells) for 48 h. The secretion of IL-10 was measured by ELISA in different siRNA-transfected-PAMs. ( C ) The secretion of IL-10 was detected in the TDG siRNA-transfected-PAMs in 0–24 h, 24–48 h, and 48–72 h after PCV2 inoculation by ELISA. The columns indicate IL-10 production in each 24 h in the culture supernatants. ( D ) The levels of IL-10 mRNA were detected in TDG siRNA-transfected-PAMs by Q-PCR with the same incubation time points as C. ( E , F ) TDG siRNA-transfected-PAMs were incubated with 5 MOI PCV2, the binding activities of NF-κB p50 and Sp1 to the il10 promoter were detected at 48 h by the ChIP assay. The data of ( A ) are representative of three independent experiments. The data of ( B – D ) are means ± SEM of three independent experiments. The data of ( E , F ) are the means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 versus NC siRNA-transfected cells in the same infection ( B , E , F ) or at the same time point ( C , D ).

Article Snippet: Cells were seeded overnight before transfection, allowed to reach 50% confluency by the time of transfection, and transfected with Akt siRNA, p38 siRNA, ERK siRNA, p50 siRNA, VG5Q siRNA, TDG siRNA, ZNF265 siRNA, and c-Myc siRNA ( ) (Sangon Biotech, Shanghai, China) using lipofectamine 2000 (Invitrogen), respectively.

Techniques: Binding Assay, Infection, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation