Journal: PLoS Pathogens

Article Title: DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA

doi: 10.1371/journal.ppat.1007742

Figure Lengend Snippet: HepAD38 cells were cultured in the presence of 2 mM PFA from day 2 to day 6 after tet removal. At day 4, the cells were re-seeded and transfected with 10 pmol siRNA targeting different cellular DNA polymerases by using RNAiMAX. (A) Total RNA was extracted at 48 h after transfection. mRNA of each indicated DNA polymerase was quantified by RT-qPCR and presented as relative amount in comparison with that in cells transfected with scramble siRNA (mean ± SD; n = 3). (B) From 48 h to 72 h after siRNA transfection (day 6 to day 7 after tet removal), cccDNA synthesis was resumed by removal of PFA from culture medium. Hirt DNA was extracted and HBV DNA was detected by Southern blot assay. The intensity of HBV cccDNA band was measured by ImageJ and presented as relative amount in comparison with that in cells transfected with scramble siRNA. Data represent 3 independent experiments (mean ± SD). Data were analyzed by two-tailed Student’s t-test (unpaired), ** P < 0.01.

Article Snippet: The Pol α1 siRNA (SR303612) and Pol α2 siRNA (SR308429) used in validation experiments ( and ) were purchased from Origene.

Techniques: Cell Culture, Transfection, Quantitative RT-PCR, Comparison, Southern Blot, Two Tailed Test

Journal: PLoS Pathogens

Article Title: DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA

doi: 10.1371/journal.ppat.1007742

Figure Lengend Snippet: (A) HepAD38 cells were cultured in the presence of 2 mM PFA from day 2 to day 6 after tet removal. At day 4, the cells were re-seeded and transfected with 10 pmol siRNA targeting POLΑ1 or scramble siRNA, by using RNAiMAX. From 48 h to 72 h after siRNA transfection (day 6 to day 7 after tet removal), PFA was removed to allow cccDNA synthesis. The cells were harvested at 72 h after siRNA transfection, and protein levels of Pol α1 and β-actin were determined by Western blot assays. Cytoplasmic HBV core DNA and Hirt DNA were extracted and detected by Southern blot assays, with mtDNA as a loading control of Hirt DNA. (B) The intensity of HBV cccDNA band was quantified by ImageJ and presented as relative amount in comparison with that in cells transfected with scramble siRNA. Data represent 6 independent experiments (mean ± SD). Data were analyzed by two-tailed Student’s t-test (unpaired). (C) HepAD38 cells were cultured in the presence of 2 mM PFA to arrest HBV replication from day 2 to day 6 after tet removal. On day 6, cccDNA synthesis was allowed by removing PFA for 24 h and treated with the indicated concentrations of CD437. Cytoplasmic HBV core DNA and Hirt DNA were extracted and detected by Southern blot assays, with mtDNA as a loading control of Hirt DNA.

Article Snippet: The Pol α1 siRNA (SR303612) and Pol α2 siRNA (SR308429) used in validation experiments ( and ) were purchased from Origene.

Techniques: Cell Culture, Transfection, Western Blot, Southern Blot, Control, Comparison, Two Tailed Test

Journal: PLoS Pathogens

Article Title: DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA

doi: 10.1371/journal.ppat.1007742

Figure Lengend Snippet: (A) Seventy-two hours after transient transfection of indicated siRNAs in HepAD38 cells, protein levels of Pol α1 and β-actin were determined by Western blot assays. (B) The mRNA levels of IL-29, CXCL10, TNF-α and IL-1β were measured by qRT-PCR and presented as fold over that in cells transfected by control siRNA (mean ± SD; n = 3). (C) HepAD38 cells were mock-treated (DMSO) or treated with 1 μM APH for 8 h. Expression of the indicated cytokine mRNA and β-actin mRNA were measured by qRT-PCR and were presented as fold over that in DMSO treated cells (mean ± SD; n = 3). (D and E) HepAD38 were cultured in the presence of 2 mM PFA from day 2 to day 6 after tet removal. From day 6, cccDNA synthesis was initiated by removing PFA for 24 h, with indicated compound treatments during the last 8 h (16 to 24 h) of cccDNA synthesis. Hirt DNA was extracted and resolved by Southern blot hybridization, with mtDNA as a loading control.

Article Snippet: The Pol α1 siRNA (SR303612) and Pol α2 siRNA (SR308429) used in validation experiments ( and ) were purchased from Origene.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Control, Expressing, Cell Culture, Southern Blot, Hybridization

Journal: PLoS Pathogens

Article Title: DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA

doi: 10.1371/journal.ppat.1007742

Figure Lengend Snippet: (A) HepAD38 cells were cultured in the presence of PFA from day 2 to day 6 after tet removal. From day 6, PFA was removed from culture media for 24 h in the presence of Pol α inhibitor APH (1 μM) or CD437 (1 μM), with DMSO as a control. Cytoplasmic HBV core DNA was extracted and analyzed by Southern blot hybridization by using HBV (-) probe to hybridize with viral plus strand. (B) HepAD38 cells were cultured in the presence of 2 mM PFA from day 2 to day 6 after tet removal. At day 4, the cells were re-seeded and transfected with 10 pmol siRNA targeting POLΑ1 or scramble siRNA, by using RNAiMAX. From 48 h to 72 h after siRNA transfection (day 6 to day 7 after tet removal), PFA was removed to resume viral DNA synthesis. Cytoplasmic HBV core DNA was extracted and analyzed by Southern blot hybridization by using HBV (-) probe to hybridize with viral plus strand DNA. (C) HepAD38 cells were cultured in the presence of PFA from day 2 to day 6 after tet removal, HBV intracellular nucleocapsids were purified by 30% sucrose cushion ultracentrifugation. In vitro EPR assay was performed at 37°C for 16 h in the absence or presence of Pol α inhibitor APH (1 μM) or CD437 (1 μM), with or without 0.1 mM of each dNTP. Upon completion of EPR reaction, viral DNA was extracted and analyzed by Southern blot hybridization. (D) HepAD38 cells were cultured in the absence of tet and HBV DNA replication was arrested by PFA treatment between day 2 to 6 after tet removal. The cells were then cultured in the presence of tet and absence of PFA for 24 h. Hirt DNA without prior treatment, treated with Exo I & III without or with following EcoRI digestion were resolved by agarose gel electrophoresis and detected by Southern blot hybridization with a minus-strand specific probe. (E) HepAD38 cells were cultured in the presence of PFA from day 2 to day 6 after tet removal. From day 6, cccDNA synthesis was initiated by removing PFA for 24 h in the presence of Pol α inhibitor APH (1 μM) or CD437 (1 μM), with DMSO as a control. Hirt DNA was extracted and analyzed by Southern blot hybridization without prior heat denaturation, or after Exo I & III digestion. Minus strand covalently closed DNA is denoted.

Article Snippet: The Pol α1 siRNA (SR303612) and Pol α2 siRNA (SR308429) used in validation experiments ( and ) were purchased from Origene.

Techniques: Cell Culture, Control, Southern Blot, Hybridization, Transfection, DNA Synthesis, Purification, In Vitro, Agarose Gel Electrophoresis

Journal: Cancer Research Communications

Article Title: The Proteogenomics of Prostate Cancer Radioresistance

doi: 10.1158/2767-9764.CRC-24-0292

Figure Lengend Snippet: Characteristics of radioresistance modulator candidates in primary prostate tumors. A, Significant associations (FDR ≤ 0.05) between CNAs in candidate genes and biochemical recurrence, as shown by fitting Cox proportional hazard models in ICGC. B, POLQ amplification is associated with biochemical relapse in ICGC (Cox proportional hazard model; FDR = 7.78 × 10 −3 ). C, High POLQ RNA abundance is associated with BCR following an RT treatment in NCCS (Cox proportional hazard model; P = 0.06). D, Increased RNA abundance of POLQ is associated with a high mutation ratio in clinically relevant candidate genes based on linear regression in ICGC. P adjusted levels after FDR correction are presented on the (right). E, Significant correlations between RNA abundances of clinically relevant candidates and POLQ RNA abundance. F and G, Strong correlation between POLQ and BRCA2 RNA abundance in ICGC ( F ) and NCCS ( G ). H, Increased RNA abundance of POLQ is associated with high Gleason grades. In F–H, ρ: Spearman correlation. P ρ : the P adjusted after FDR.

Article Snippet: To achieve POLQ knockdown using siRNA degradation, parental, CF-resistant, and HF-resistant DU145 cells were seeded in six-well plates, and 24 hours later, cells were transfected with scrambled siRNA (control; CAT. #: SR30004; Origene Inc.) or POLQ siRNA (CAT. #: SR323222; Origene Inc.) using siTran 2.0 Transfection Reagent (CAT. #: TT320001; Origene Inc.) and left overnight for at least 24 hours before performing experiments.

Techniques: Amplification, Mutagenesis

Journal: Cancer Research Communications

Article Title: The Proteogenomics of Prostate Cancer Radioresistance

doi: 10.1158/2767-9764.CRC-24-0292

Figure Lengend Snippet: The proteome signature upon POLQ inhibition. A, Experimental design schematic. B, Genetic POLQ inhibition. The parental, CF-resistant, and HF-resistant cells were treated with either POLQ siRNA or scramble siRNA as the control. Significant radiosensitization was observed in all cell lines upon POLQ knockdown ( P = 0.01, 0.002, and 0.02 for the parental, CF-resistant, and HF-resistant cells, respectively; paired t test). Three biological replicas of 4,000 cells per well and three of 6,000 cells per well were considered for each sample. C, Pharmacologic POLQ inhibition. CF-resistant, HF-resistant, and the parental cells were treated with 100 μmol/L novobiocin to achieve POLQ inhibition (Nvb 100 µmol/L). Significant radiosensitization was observed in all DU145 cell lines upon POLQ inhibition ( P = 0.04, 0.02 and 0.01, for the parental, CF-resistant, and HF-resistant cells; paired t test). In B and C, both the control and the treated cells were irradiated with 0 or 4 Gy in two fractions. The surviving fraction of the treated cells ( POLQ siRNA or Nvb 100 µmol/L) was normalized to the surviving fraction of the corresponding control of each cell line (black dots). D, The (top) 10 activated and suppressed biological processes at the protein level following POLQ depletion, in CF-resistant cells and the parental. The dot size represents the enrichment score, and the dot color is the directionality: orange shows upregulation toward POLQ -treated cells. E, POLQ genetic inhibition creates a proteomic signature, involving 12 affected genes in CF-resistant cells. Three replicates from each cell type were used for the analysis. Left: signature genes whose protein abundances were changed between POLQ -depleted cells and the control, in CF-resistant cells and the parental cells. The dot size represents the Cohen’s d effect size, and the dot color is the directionality: magenta, upregulation, and green, downregulation toward POLQ -treated cells. Middle: changes in protein abundances of signature genes, between CF-resistant cells and the parental. The dot size represents the Cohen’s d effect size, and the dot color is the directionality: magenta, upregulation, and green, downregulation toward CF-resistant cells. Right: changes in RNA abundances of signature genes between CF-resistant cells and the parental. The dot size represents the log 2 (fold change) values. The dot color represents the directionality: red shows upregulation toward CF-resistant cells. F, Investigating signature genes (that were affected by POLQ inhibition in cell lines) in primary patient data. Left: RNA-RNA Pearson correlations between POLQ and signature genes. Middle, Pearson correlation between the abundances of POLQ RNA and proteins of signature genes. Right: Abundance of signature genes in normal vs. tumor samples at the RNA and protein levels. For all panels, datasets refer to the cohort names used for analysis and the data type for the type of molecule tested for signature genes (i.e., RNA or protein).

Article Snippet: To achieve POLQ knockdown using siRNA degradation, parental, CF-resistant, and HF-resistant DU145 cells were seeded in six-well plates, and 24 hours later, cells were transfected with scrambled siRNA (control; CAT. #: SR30004; Origene Inc.) or POLQ siRNA (CAT. #: SR323222; Origene Inc.) using siTran 2.0 Transfection Reagent (CAT. #: TT320001; Origene Inc.) and left overnight for at least 24 hours before performing experiments.

Techniques: Inhibition, Control, Knockdown, Irradiation

Journal: Scientific reports

Article Title: Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells.

doi: 10.1038/s41598-018-19216-1

Figure Lengend Snippet: Figure 3. Damaged mitochondrial DNA activates ZBP1/TBK1/IRF3 signaling pathway. Interaction between BrDU- labelled mtDNA and TFAM (A) and BrDU-labelled mtDNA and ZBP1 (B) in control and GOx-treated cells at 1 h. (C) Interaction between ZBP1/TBK1, IRF3/TBK1, ZBP1/P-Tyr and ZBP1/P-Ser in control and GOx-treated cells at 1 h. (D) Interaction between IRF3/TBK1 in pre-treated with 3 μM of CsA in GOx-treated BEAS 2B cells. (E) Level of ZBP1 depletion, shown by Western blotting. (F) Expression of IL-6 and IL-8 in unstressed and GOx-treated (0.006 U/ ml for 1 h) in control and ZBP1-depleted BEAS2B cells. Representative images of n = 3 independent experiments are shown. Data represent average ± SEM of n = 5 biological replicates. *p < 0.05 vs. control.

Article Snippet: For transient ZBP1 depletion we used siRNA construct specific for human ZBP1 (Origene, SR312989B).

Techniques: Control, Western Blot, Expressing

Journal: Scientific reports

Article Title: Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells.

doi: 10.1038/s41598-018-19216-1

Figure Lengend Snippet: Figure 5. Inflammation respond to mitochondrial DNA in vivo. (A) Inflammatory response triggered in BALF of naïve mice at 24 h post intratracheal administration of 1 μg of isolated mtDNA. (B) Time-dependent expression of IL-1α in BEAS 2B cells incubated 100 ng of isolated mtDNA. (C) Model of inflammation induced by mtDNA/ZBP1 pathway. Data represent average ± SEM using n = 6–8 animals or n = 3 for biological replicates per experimental end-point. *p < 0.05, **p < 0.01 vs. control.

Article Snippet: For transient ZBP1 depletion we used siRNA construct specific for human ZBP1 (Origene, SR312989B).

Techniques: In Vivo, Isolation, Expressing, Incubation, Control