Journal: BMC Biochemistry

Article Title: Human POLD1 modulates cell cycle progression and DNA damage repair

doi: 10.1186/s12858-015-0044-7

Figure Lengend Snippet: Modulation of POLD1 expression in HEK293 cells. a qRT-PCR analysis of POLD1 mRNA level in HEK293 cells transfected with pcDNA3.0-POLD1, pcDNA3.0, shPOLD1 or shControl. b Western blot analysis of POLD1 protein level in HEK293 cells transfected with pcDNA3.0-POLD1, pcDNA3.0, shPOLD1 or shControl. Untransfected HEK293 cells were used as a control (Blank). n = 3, * P < 0.01 vs. the negative control (pcDNA3.0 or shControl)

Article Snippet: Short hairpin RNA (shRNA) targeting POLD1 (shPOLD1) and the negative control shRNA (shControl) were purchased from BGI (Shenzhen, China).

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

Journal: BMC Biochemistry

Article Title: Human POLD1 modulates cell cycle progression and DNA damage repair

doi: 10.1186/s12858-015-0044-7

Figure Lengend Snippet: Effects of altered POLD1 expression on the growth of HEK293 cells. HEK293 cells were transfected with the indicated vectors, and cell proliferation was determined by CCK-8 assay 24, 48, and 72 h later. Data shown were the mean ± SD of the ratio for the absorbance at 450 nm. n = 3, * P < 0.01 vs. shControl

Article Snippet: Short hairpin RNA (shRNA) targeting POLD1 (shPOLD1) and the negative control shRNA (shControl) were purchased from BGI (Shenzhen, China).

Techniques: Expressing, Transfection, CCK-8 Assay

Journal: BMC Biochemistry

Article Title: Human POLD1 modulates cell cycle progression and DNA damage repair

doi: 10.1186/s12858-015-0044-7

Figure Lengend Snippet: Effects of altered POLD1 expression on e cell cycle progression of HEK293 cells. a Representative FACS plots showing cell cycle distribution of HEK293 cells transfected with the indicated vectors. b Quantification of the percentage of cells in different phases. n = 3, * P < 0.01 vs. shControl

Article Snippet: Short hairpin RNA (shRNA) targeting POLD1 (shPOLD1) and the negative control shRNA (shControl) were purchased from BGI (Shenzhen, China).

Techniques: Expressing, Transfection

Journal: BMC Biochemistry

Article Title: Human POLD1 modulates cell cycle progression and DNA damage repair

doi: 10.1186/s12858-015-0044-7

Figure Lengend Snippet: Quantification of DNA synthesis rate in HEK293 cells. a HEK293 cells were transfected with the indicated vectors, 48 h after transfection EdU-positive cells were determined by flow cytometry. b Quantification of the percentage of cells positive for EdU incorporation assay shown in ( a ). n = 3, * P < 0.01 vs. shControl

Article Snippet: Short hairpin RNA (shRNA) targeting POLD1 (shPOLD1) and the negative control shRNA (shControl) were purchased from BGI (Shenzhen, China).

Techniques: DNA Synthesis, Transfection, Flow Cytometry

Journal: BMC Biochemistry

Article Title: Human POLD1 modulates cell cycle progression and DNA damage repair

doi: 10.1186/s12858-015-0044-7

Figure Lengend Snippet: Effects of altered POLD1 expression on DNA damage in HEK293 cells. a HEK293 cells were transfected with the indicated vectors and treated with 150 μM H 2 O 2 for 5 min in the dark. Comet assay was then performed, and images were acquired using a fluorescence microscope to show DNA fragment migration patterns. b Quantification of the tail moments from the comet assay shown in ( a ). * P < 0.01, vs. the negative control (shControl or shControl + H 2 O 2 )

Article Snippet: Short hairpin RNA (shRNA) targeting POLD1 (shPOLD1) and the negative control shRNA (shControl) were purchased from BGI (Shenzhen, China).

Techniques: Expressing, Transfection, Single Cell Gel Electrophoresis, Fluorescence, Microscopy, Migration, Negative Control

Journal: Annals of Translational Medicine

Article Title: Macrophage regulator of G-protein signaling 12 contributes to inflammatory pain hypersensitivity

doi: 10.21037/atm-20-5729

Figure Lengend Snippet: RGS12 can strengthen the transcriptional regulation of COX2 via NF-κB. (A) Western blot analysis of RGS12 expression. Primary BMMs were transfected with pCMV-empty (control) or pCMV-RGS12 (RGS12 OE) plasmids by Lipofectamine 3000 for 24 hours, and the expression levels of RGS12 were analyzed. **, P<0.01 vs. the control group, n=3. (B) Relative mRNA expression of COX2 was analyzed by real-time PCR. Primary BMMs were treated as described in (A), and the expression levels of COX2 were analyzed. **, P<0.01, ***, P<0.001 compared with the control group. The values are the mean ± SEM (n=5). (C) COX2 expression levels in BMMs were analyzed by ELISA. Primary BMMs were treated as described in (A), and COX2 expression levels were analyzed. Note that RGS12 OE enhanced the expression level of COX2. **, P<0.01, ***, P<0.001 compared with the control group. (D) Relative protein levels of NF-κBp65. RAW264.7 cells were transfected with shControl (pSicoR-empty) or shNF-κB plasmids (pSicoR-shNFκBp65-1 and pSicoR-shNFκBp65-2) for 24 hours. β-Actin was used as an internal reference. The data are presented as the means ± SEM. ***, P<0.001 vs. shControl group, n=3. (E) Western blot analysis of NF-κBp65. RAW264.7 cells were transfected with pcDNA3.1-empty (control) or pcDNA3.1-NF-κBp65 (NF-κBp65 OE) plasmids for 24 hours. β-Actin was used as an internal reference. The data are presented as the means ± SEM. ***, P<0.001 vs. the control group, n=3. (F) RAW 264.7 cells were transfected with pUC-shNF-κBp65 and/or pCMV-RGS12 for 24 hours and then induced with TNFα for 24 hours. Relative mRNA expression was measured by real-time PCR. Note that RGS12 could not increase COX2 expression when NF-κB expression was decreased. The values are the mean ± SEM. ***, P<0.001, n=5. (G) Analysis of the interactions between RGS12-PTB and NF-κBp65 by pulldown assay. RAW264.7 cells were transfected with pCMV-Flag or pCMV-PTB-Flag plasmids, and cell lysates were precipitated with anti-Flag affinity gel and immunoblotted (IB) with anti-NF-κB p65 and anti-Flag antibodies. (H) RGS12 promotes the transcriptional activity of NF-κB and COX2 expression (luciferase assay). Stable knockdown of NF-κB in RAW264.7 cells was achieved via the transfection of pSicoR-shNFκBp65-1 and pSicoR-shNFκBp65-2 vectors. The stably transfected RAW264.7 cells were treated with COX2-Luc, pcDNA3.1-NF-κBp65, pCMV-RGS12 and/or pCMV-PTB for 48 hours. *, P<0.05, ***, P<0.001 versus the control. The data are presented as the means ± SEM. Note that RGS12 or PTB could increase the transcriptional activity of NF-κB.

Article Snippet: RAW264.7 cells were transfected with shControl (pSicoR-empty) or shNF-κB plasmids (pSicoR-shNFκBp65-1 and pSicoR-shNFκBp65-2) for 24 hours. β-Actin was used as an internal reference.

Techniques: Western Blot, Expressing, Transfection, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activity Assay, Luciferase, Knockdown, Stable Transfection