EMBO/soda-nel huggingface dataset:
PWWP2B promotes DNA End Resection and Homologous Recombination (2022)
Figure 4

Figure 4. PWWP2B is implicated in DDR and HR-mediated DSB repair.

A. The numbers of γH2AX foci at the indicated time points after exposure to Gy of ionizing radiation (IR) in HeLa cells transfected with either control or PWWP2B siRNA1 or 2. The results represent the average of three independent experiments.The error bars indicate the standard deviation for cells transfected with each siRNA.

B-C. Measurement of homologous recombination and non-homologous end-joining capacity in DR-GFP and EJ5-GFP reporter U2OS cells. (B) U2OS cells harboring the DR-GFP reporter were treated with the indicated siRNAs, followed by transfection with the I-SCEI expression vector. Two days later, GFP expression was accessed by flow cytometry. BRCA depletion was used as a negative control. (C) The NHEJ assay was performed with U2OS cells harboring the NHEJ reporter (EJ5-GFP). The reporter cells were transfected with the indicated siRNAs, followed by transfection with the I-SCE1 expression vector. Two days later, the GFP-expressing cells were accessed by flow cytometry. LIG4 depletion was used as a negative control. The GFP expression levels are shown as the average of three independent experiments, and the error bars indicate the standard deviation.

D-G. Quantification of radiation-induced RAD51, BRCA1, and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs. All data were obtained from over cells (open circles), and the mean values are shown as red bars (upper panel). Immunofluorescence analysis of irradiation-induced RAD51, BRCA1, and 53BP1 foci formation. HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (D and E), BRCA1 (F), or 53BP1 (G) antibodies. The cells in S phase were identified by Cyclin A staining. (E) The siPWWP2B-resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA-mediated PWWP2B depletion. IR-induced RAD51 foci formation was examined by immunofluorescence.

H. Cell survival of the PWWP2B-depleted HeLa cells in response to IR. HeLa cells were transfected with either control or PWWP2B siRNAs. After 48 h of transfection, 500 cells were plated and irradiated with the indicated doses of IR. The number of surviving colonies was counted 14 days after IR exposure. These experiments were performed in triplicate, and the results represent the average of three independent experiments. The error bars indicate the standard deviation.
PWWP2B promotes DNA End Resection and Homologous Recombination (2022)
Figure 5

Figure 5. PWWP2B participates in DNA end resection during HR.

A. Immunoblot analysis of RAD51, BRCA1, or 53BP1 protein levels in PWWP2B-depleted HeLa cells.

B. RPA phosphorylation at the indicated time points in response to IR exposure. HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4/S8 phosphorylation and total RPA.

C-D. Quantification of radiation-induced RPA2 or S4/S8 phosphorylated RPA foci formation in PWWP2B-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RPA2 or S4/S8 phosphorylated RPA2 foci formation. HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RPA2 (C), or S4/S8 phosphorylation-specific RPA2 (D) antibodies (bottom panel).

E. Schematic illustration of the quantitation of DNA resection in human cells by the ER-AsiSI assay. The AsiSI site located on chromosome 1 is indicated as a red bar. Three pairs of qPCR primers were designed crossing the BsrGI restriction sites and the distances from the AsiSI sites are written as numbers.

F. ER-AsiSI U2OS cells were transfected with the indicated siRNAs. Two days after transfection, the cells were treated with 300 nM 4-OHT for 4 h, then the genomic DNA from those cells was extracted and digested with BsrGI overnight. DNA end resection near the AsiSI site was measured by qPCR as described in the Materials and Methods. The results are the average of three experiments and the error bars indicate standard deviations.
PWWP2B promotes DNA End Resection and Homologous Recombination (2022)
Figure 6

Figure 6. PWWP2B is associated with MRE11.

A. Immunofluorescence analysis for irradiation-induced MRE11 foci formation (left panel). HeLa cells transfected with either control siRNA, siPWWP2B or siUHRF1 were irradiated, fixed and subjected to staining with MRE11 and γH2AX antibodies. More than 100 nuclei per condition were counted.The percentage of MRE11-positive cells is presented as a bar graph (right panel). The results represent the average of three independent experiments. The error bars indicate standard deviation.

B. HeLa cells expressing GFP-MRE11 were treated with control siRNA or siRNAs against PWWP2B. After 48 h, the cells were subjected to laser microirradiation. Stripes on the laser were examined at the indicated time points (upper panel).The intensity of the laser stripes at the time points was determined. The average intensity values from 10 cells were graphed in the bottom panel.

C. The interaction between PWWP2B and MRE11. HA-PWWP2B and Myc-MRE11-expressing vectors were transfected into 293T cells. After 24 h, the cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblot analysis with the indicated antibodies.

D. The interaction between endogenous PWWP2B and MRE11. Immunoprecipitation reactions were performed using rabbit IgG or anti-MRE11 antibodies, followed by immunoblot analysis with the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.

E. HA-MRE11 and either GFP-PWWP2B WT or its serial deletion mutants were co-transfected into 293T cells. The cell lysates were immunoprecipitated with anti-HA antibody and then immunoblotted with the indicated antibodies.
PWWP2B promotes DNA End Resection and Homologous Recombination (2022)
Figure 7

Figure 7. PWWP2B and UHRF1 are epistatic in DDR and HR-mediated DSB repair.

A. Comparison of IR-induced γH2AX foci formation in HeLa cells treated with the indicated siRNAs.

B-C. Comparison of IR-induced foci formation for either RAD51 or BRCA1 in HeLa cells transfected with the indicated siRNAs. Quantification of radiation-induced RAD51 or BRCA1 foci formation in PWWP2B alone, UHRF1 alone or PWWP2B and UHRF1-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RAD51 or BRCA1 foci formation. HeLa cells treated with the indicated siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (B), or BRCA1 (C) antibodies (bottom panel).

D. Comparison of cell sensitivity to IR. HeLa cells transfected with indicated siRNA (N = 500) were plated and treated with the indicated doses of IR. The number of surviving colonies was counted 14 days after IR treatment. The average of three independent experiments is shown. The error bars indicate the standard deviation.

E. Comparison of homologous recombination repair capacity in DR-GFP U2OS cells transfected with the indicated siRNAs.
The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection (2018)
Figure 1

Figure 1: The MCMV m152 protein specifically targets STING-dependent signaling

(A) 293T cells were co-transfected with expression plasmids for Cherry-STING, the murine IFNβ-luciferase reporter (IFNβ-Luc), a Renilla luciferase normalization control (pRL-TK) and the indicated expression plasmids or empty vector (ev). Cells were additionally co-transfected with expression plasmids for cGAS-GFP (stimulated) or IRES-GFP (unstimulated). 20 hours post transfection, cells were lysed and a dual-luciferase assay was performed.

(B) An expression plasmid for RIG-I N (stimulated) or ev (unstimulated) was co-transfected with IFNβ-Luc, pRL-TK and the indicated expression plasmids in 293T cells and analyzed as in (A).

(C) An expression plasmid for TBK1 (stimulated) or ev (unstimulated) was co-transfected together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A).

(D) 293T cells were co-transfected with a plasmid expressing constitutively active IRF3 (IRF3-5D; stimulated) or IRES-GFP (unstimulated) together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A).

(E) The ISG56-luciferase reporter, pRL-TK and the indicated expression plasmids were co-transfected in 293T cells. 24 hours post transfection, cells were stimulated with 0.1 ng/µl human IFNβ or mock stimulated and analyzed 16 hours later as described in (A).

(F-G) iMEFgt/gt stably expressing Cherry-STING and either ev or V5-tagged m152 were stimulated with 5 µg/ml ISD (F), 10 µg/ml poly(I:C) (G) or mock stimulated with Lipofectamine. 4 hours post stimulation, RNA was extracted to determine IFNβ mRNA transcripts by qRT-PCR.

(H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 10 µg/ml cGAMP (H), 5 µg/ml ISD (I), Newcastle's Disease Virus (NDV) infection (J) or 1 µM CpG DNA (K). 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) or TNFα (K) levels were determined by ELISA.

Data information: (A-G) Data is combined from three independent experiments. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown.

Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.
The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection (2018)
Figure 2

Figure 2: STING and the MCMV m152 protein colocalize and interact under unstimulated and stimulated conditions

(A-B) (A) HeLa cells were co-transfected with expression plasmids for Cherry-STING, V5-tagged m152 and either ev (unstimulated) or cGAS-GFP (stimulated).

(B) iMEFgt/gt were co-transfected with expression plasmids for V5-tagged m152 together with either Cherry-STING or ev in combination with cGAS-GFP (stimulated) or ev (unstimulated). 24 hours post transfection cells were fixed for immunolabeling with an anti-V5 antibody. White boxes indicate the region shown at a higher magnification. Scale bar represents 10 µm.

(C) Lysates of Cherry-STING and either CD4-V5 or m152-V5 expressing 293T cells were subjected to immunoprecipitation (IP) with an anti-V5 antibody. Input and IP samples were analyzed by IB with the indicated antibodies.

(D) iMEF stably expressing ev or m152-V5 were left unstimulated or stimulated with 10 µg/ml ISD and lysed 90 minutes later. m152 was immunoprecipitated with an anti-V5 antibody and samples were analyzed by IB with V5, STING and phospho-TBK1 (pTBK1) specific antibodies.

Data information: IB shown are representative of three (C) or two (D) independent experiments.
The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection (2018)
Figure 3

Figure 3: The N-terminal domain of m152 directs the interaction with STING

(A) Schematic representation of wildtype m152, wildtype CD4, CD4-m152 chimeric constructs, m152 glycosylation mutants, and m152 stalk mutants used in this study. CD4-m152 chimeras (1-6): The relevant domain of m152 was replaced singly or in combination with the respective domain of murine CD4. m152 glycosylation mutant (7): Asparagine (N) at position 83, 230 and 263 were mutated to Glutamine (Q). m152 stalk mutants (8-9): m152-Δstalk has a deletion of amino acids 300-326 (8), and for m152-GSstalk a glycine-serine linker ((G4S)5) was inserted to replace the stalk region (9). SP = signal peptide, NTD = N-terminal domain, TM = transmembrane domain, CTD = C-terminal domain. Branched symbols represent the three glycosylation sites of m152.

(B) Cherry-STING, IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids as shown in (A) were transiently expressed in 293T cells and a dual luciferase assay was performed. Data is combined from two out of three independent experiments and shown as mean ± SD.

(C) 293T cells were co-transfected with expression plasmids for Cherry-STING and indicated chimeras as shown in (A). An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. IB shown is representative of three independent experiments.

(D) Cherry-STING, IFNβ-Luc, pRL-TK and either CD4, m152, m152-Δstalk or m152-GSstalk were transiently expressed in 293T cells. For stimulation, samples were co-transfected with cGAS-GFP, and unstimulated samples with IRES-GFP. Lysates were analyzed as described in (B). Data is combined from two out of three independent experiments and shown as mean ± SD.

(E) 293T cells were co-transfected with expression plasmids for Cherry-STING and either CD4, m152, m152-Δstalk or m152-GSstalk. An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. Immunoblot shown is representative of three independent experiments.

Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001
The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection (2018)
Figure 4

Figure 4: Binding of m152 to both ER luminal loop regions of murine STING is a prerequisite for its antagonistic activity

(A) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids together with murine Cherry-STING (left panel) or human STING (right panel). Lysates were analyzed by dual-luciferase assay. Data is combined from two out of three independent experiments and shown as mean ± SD.

(B) Schematic representation of the predicted m152 (red) and STING (grey) topology in the ER membrane. (1) and (2) specify the ER luminal loop regions of STING with the sequence alignments from murine and human STING shown below. In murine STING, N41 in loop (1) was mutated to E41 and loop (2) was exchanged with loop (2) of human STING (hL2). The resultant constructs were designated mSTING N41E, mSTING hL2 and mSTING N41E-hL2. In human STING, mutations were introduced vice versa, resulting in hSTING E41N, hSTING mL2 and hSTING E41N-mL2.

(C) IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either LacZ or m152 together with the indicated murine STING mutants described in (B) were transiently expressed in 293T cells. Luciferase activity was measured as described in (A).

(D) 293T cells were co-transfected with expression plasmids for either LacZ or m152 together with the indicated murine STING mutants described in (B). An anti-V5 IP was performed and samples were analyzed by IB with the indicated antibodies.

(E-F) Luciferase assay (E) and co-IP (F) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B).

Data information: Data in (C) and (E) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD. IB shown in (D) and (F) are representative of three independent experiments.

Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001
The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection (2018)
Figure 5

Figure 5: m152 antagonizes STING translocation, but not its activation by cGAMP or its dimerization

(A) Schematic representation of the key steps in the cGAS-STING signaling pathway.

(B) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK and either ev or m152. Cells were further co-transfected with either Cherry-STING WT or the constitutively active Cherry-STING V154M (stimulated) or with IRES-GFP (unstimulated). Data is combined from three independent experiments and shown as mean ± SD.

(C) iMEF stably expressing ev or m152-V5 were stimulated with 10 µg/ml ISD for the indicated times or left unstimulated (mock). Cell lysates were analyzed by SDS-PAGE under non-reducing conditions and subjected to IB with the specified antibodies.

(D) Two representative still images from live cell imaging experiments with iMEFgt/gt stably expressing Cherry-STING transfected with ISD (right panel) or left unstimulated (left panel). Red circles highlight representative translocated STING in ISD stimulated cells, which is used as an indicator of activation.

(E) iMEFgt/gt stably expressing Cherry-STING and either ev or m152-V5 were stimulated with ISD. Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation. Data shown is one representative of two independent experiments.

(F) iMEFgt/gt stably expressing Cherry-STING and V5-tagged m152 or corresponding ev were stimulated with 5 µg/ml ISD for the indicated time or left unstimulated (mock). Lysates were subjected to IB with the specified antibodies.

Data information: IB shown in (C) and (F) are representative of three and two independent experiments, respectively. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, ****p<0.0001
The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection (2018)
Figure 6

Figure 6: MCMV lacking m152 induces an elevated type I IFN response leading to lower levels of viral transcripts in vitro

(A) Schematic representation of the recombinant MCMV m152stop virus constructed on the MCMV Δm157 backbone (here referred to as parental MCMV and MCMV m152stop for simplicity). Shown is the transcriptional coding region of m152. STOP indicates the introduced 16 basepair (bp) stop cassette.

(B) iMEF were infected by centrifugal enhancement with either parental MCMV (par.) or MCMV m152stop at an MOI of 0.5 or mock infected. 3 hours post infection (hpi) cells were lysed and subjected to immunoblotting (IB) with the specified antibodies.

(C) iMEF and iMEFgt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop, or parental MCMV alone, respectively (MOI 0.5). 3 hpi lysates were subjected to an anti-m152 IP and samples were analyzed by IB with indicated antibodies. IB shown is representative of two independent experiments.

(D) Primary BMDM were infected with parental MCMV (par.) or MCMV m152stop at an MOI of 0.1 or mock infected. 16 hpi secreted levels of IFNα or IFNβ were quantified by ELISA. Data is representative of two independent experiments.

(E) iMEFgt/gt stably expressing Cherry-STING were infected with parental MCMV or MCMV m152stop (MOI 0.5). Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation. Data shown is one representative of two independent experiments.

(F-G) iMEF or iMEFgt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). At 6 hpi total RNA was extracted to determine MCMV IE1 and MCMV E1 (F), IFNb1 and IL6 (G) transcripts by qRT-PCR. Data shown is combined from two out of three independent experiments.

(H) 293T cells were co-transfected with Cherry-STING, the pNF-κB luciferase reporter, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either ev or m152. Cells were lysed and analyzed as described in Figure 1. Data is combined from three independent experiments.

Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01. Data are shown as mean ± SD.
The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection (2018)
Figure 7

Figure 7: In the absence of STING the impairment of MCMV m152stop in vivo is rescued

(A-C) Type I IFN levels in spleen homogenates (A) and serum (B) and IL6 levels in spleen homogenates (C) of C57BL/6J (B6J) or STING-/- mice following i.v. infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA. IFNβ levels of STING-/- mice were below the detection limit (n.d.).

(D) B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV or MCMV m152stop. 6 hpi, RNA was extracted from spleen homogenates and expression of MCMV IE1 and E1 transcripts was determined by qRT-PCR.

(E) B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV. 6 hpi, RNA was extracted from spleen homogenates and m152 transcript levels were determined by qRT-PCR.

(F) B6J or STING-/- mice were depleted of NK cells via treatment with anti-NK1.1 (right panel) or left untreated (left panel). One day after NK cell depletion, B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV or MCMV m152stop. 16 hpi, RNA was extracted from spleen homogenates and MCMV E1 transcript levels were determined by qRT-PCR. Data were normalized to 107 cellular β-actin transcripts (D-F).

Data information: n = 5-6 mice per group; Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001
The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection (2018)
Figure 8

Figure 8: The STING-mediated NF-κB response is activated from the ER and specifically pro-viral for MCMV transcription

(A) 293T cells were co-transfected with Cherry-STING, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either the pNF-κB, p55-CIB, p125 or p125AA luciferase reporter. Data is representative of two independent experiments.

(B) 293T cells were co-transfected with expression plasmids for cGAS-GFP (stimulated) or ev (unstimulated) together with either cherry-tagged WT STING or cherry-tagged K288R STING. Cells were fixed for imaging 24 hours post transfection. Scale bar represents 10 µm.

(C-D) iMEFgt/gt stably expressing cherry-tagged WT STING or K288R STING and either ev or V5-tagged m152 were stimulated with ISD (10 µg/ml) or mock stimulated. At 4 hpi total RNA was extracted to determine IFNb1 (C) and IL6 (D) transcripts by qRT-PCR. Data shown is combined from three (C) or two (D) out of three independent experiments.

(E) iMEFgt/gt (-) and iMEFgt/gt stably expressing either cherry-tagged WT STING or K288R STING were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). 6 hpi total RNA was extracted to determine MCMV E1 transcripts by qRT-PCR. Data shown is combined from three independent experiments.

Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0,05, **p<0.01, ****p<0.0001. Data are shown as mean ± SD.