Journal: Frontiers in Immunology

Article Title: Deciphering a mitochondria-related signature to supervise prognosis and immunotherapy in hepatocellular carcinoma

doi: 10.3389/fimmu.2022.1070593

Figure Lengend Snippet: Verification of the expression of six mitochondrial-related genes by immunohistochemistry and qRT-PCR analysis. (A–F) The protein expression of six mitochondrial-related genes in HCC and adjacent normal tissues by immunohistochemistry. (A) MTHFD1L. (B) NT5DC2. (C) RECQL4. (D) TOMM40L. (E) TXNRD1. (F) POLQ. (G–R) The mRNA expression of six mitochondrial-related genes in HCC tissues and cell lines by qRT-PCR analysis. (G, M) MTHFD1L. (H,N) NT5DC2. (I, Q) POLQ. (J, P) RECQL4. (K, Q) TOMM40L. (L, R) TXNRD1. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The regents and citations are as follows: MTHFD1L: Atlas Antibodies Cat#HPA029041, RRID: AB_2672880, dilution: 1:350; NT5DC2: Atlas Antibodies Cat#HPA050683, RRID: AB_2681215, dilution: 1:40; RECQL4: Atlas Antibodies Cat#HPA025821, RRID: AB_1856168, dilution: 1:40; TOMM40L: Atlas Antibodies Cat#HPA051304, RRID: AB_2681432, dilution: 1:75; TXNRD1: Santa Cruz Biotechnology Cat#sc-28321, RRID: AB_628405, dilution: 1:2500; POLQ: Atlas Antibodies Cat#HPA026762, RRID: AB_10601248, dilution: 1:450.

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A Endogenous RecQL4 or RNF8 were detected in anti-Flag immunoprecipitated fractions from Flag-RNF8/Flag-RecQL4 transfected U2OS cells by western blotting analysis. Flag-GFP/Empty vector-transfected cells were used as negative controls. B Interaction between endogenous RecQL4 and RNF8. Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz). RecQL4 protein was detected in the RNF8 immunoprecipitated complex. C Direct interaction between RecQL4 and RNF8 was demonstrated by an in vitro pull-down assay. Purified recombinant GST-RNF8 was immobilized on Glutathione resin and incubated with purified Flag-RecQL4 in the IP buffer, and the bound proteins were examined by western blotting. D Colocalization of GFP-tagged 53BP1 and mCherry-tagged RNF8/RecQL4 at DSB track induced by UV micro-point laser. E Co-localization of mCherry-RNF8 and GFP-RecQL4 at DSB track induced by UV micro-point laser. F Co-localization of endogenous RNF8 and RecQL4 after X-ray irradiation. U2OS cells were exposed to 10 Gy of X-ray irradiation (25 mA, 160 kV; dose rate 0.995 Gy/min, X-RAD RS2000, Rad Source, USA), and fixed with 4% paraformaldehyde at 3.5 h post treatment. Indirect immunostaining was performed using primary anti-RecQL4 and RNF8 and fluorescence-dye conjugated secondary antibodies. After counterstaining with DAPI, images were captured using a fluorescence microscope (Leica DM5000 Microsystems).

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation, In Vitro, Pull Down Assay, Purification, Recombinant, Incubation, Irradiation, Immunostaining, Fluorescence, Microscopy

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A RecQL4 ubiquitination level was decreased upon RNF8 depletion. U2OS cells were transfected with Flag-RecQL4 plasmid, followed by control/RNF8 shRNA and HA-Ub plasmids. Immunoprecipitation was performed on the cell lysates using anti-Flag (M2) beads, and RecQL4 ubiquitination level was determined by western blotting analysis with anti-HA antibody. B RNF8 promotes RecQL4 ubiquitination in vivo. U2OS cells were transfected with Flag-RecQL4, HA-Ub, and RNF8 plasmids. Empty vector was used as control. C , D RecQL4 CT mutant (3M: K876R-K2048R-K1101R) showed a markedly decreased ubiquitination level relative to wild-type RecQL4 CT in both in vivo and in vitro ubiquitination assays.

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Control, shRNA, Immunoprecipitation, Western Blot, In Vivo, Mutagenesis, In Vitro

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A Knockdown of RNF8 significantly inhibits the dissociation of RecQL4 from DSBs. U2OS cells with or without RNF8 depletion were treated with 365-nm micro-point laser. The recruitment of GFP-RecQL4 at DSBs was recorded and the fluorescence density was quantified. B The prolonged and enhanced recruitment of RecQL4-3M at DSBs induced by UV micro-point laser. U2OS cells were transfected with GFP-tagged WT RecQL4 or its 3M mutant, and subjected to UV micro-point laser treatment. The recruitment of GFP-RecQL4 or its mutant at DSBs were recorded and the fluorescence density at DSBs was quantified. At least 15 cells were analyzed for each treatment. The data represent mean ± SEM from three independent experiments. ** p < 0.01.

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Knockdown, Fluorescence, Transfection, Mutagenesis

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A , B RecQL4 depletion significantly decreased the HR- and NHEJ-mediated DSB repair in U2OS cells quantified by DR-GFP and EJ5-GFP reporter system, respectively. The defective DSB repair was significantly restored by re-introduction of wild-type RecQL4 but not its mutant. The percentage of GFP-positive cells was quantified by Flow cytometry. The data represent mean ± SEM from three independent experiments. (* p < 0.05, Student’s t test). C Analysis of the time-dependent recruitment of various DSB repair proteins in U2OS cells after micro-point laser treatment. Cells were transfected with GFP-tagged RecQL4, RNF8, Ku80, 53BP1, or mCherry-tagged MDC1 plasmids, followed by the treatment of micro-point laser. The images were captured using time-lapse microscopy. At least 15 cells for each transfection were recorded and analyzed for the earliest time point of protein aggregate formation at DSB track. D RecQL4 ubiquitination status affects the recruitment of its directly associated downstream protein-CtIP. RecQL4 was first silenced in U2OS cells followed by transfection with GFP-tagged CtIP and mCherry-tagged wild type RecQL4 or its mutant (3M). The mCherry-positive cells were treated with micro-point laser and the images were captured using microscopy. Both recruitment time and fluorescence density were recorded and at least 15 cells were analyzed. The data represent mean ± SEM from three independent experiments. ** p < 0.01. E RecQL4 3M mutant interferes with its capacity in processing ssDNA formation at DSB ends estimated by an end resection assay. RecQL4 was first silenced by shRNA infection in U2OS cells which were then transfected with either a control, RecQL4 WT, or 3M mutant for 24 h, followed by the treatment with 1 μM CPT (C9911, Sigma) for 1 h. Cells were fixed for RPA2 (ab2175, Abcam) immunostaining. A total of 200 cells were analyzed and RPA2-foci (>15) positive cells were scored for each individual experiment. Data represent mean ± SEM of three independent experiments (** p < 0.01, the Single Factor Anova test). Fluorescence images were captured using a LEICA TCS SP8 confocal microscope system. Scale bar, 22 μm.

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Mutagenesis, Flow Cytometry, Transfection, Time-lapse Microscopy, Ubiquitin Proteomics, Microscopy, Fluorescence, Resection Assay, shRNA, Infection, Control, Immunostaining

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A RecQL4 interacts with WRAP53β. Endogenous WRAP53β was present in Flag immunoprecipitated fractions using the lysate of HEK293 cells transfected with Flag-RecQL4. Direct interaction between RecQL4 and WRAP53β was further demonstrated by an in vitro pull-down assay using the purified recombinant GST-WRAP53β to pull down Flag-RecQL4. B A markedly enhanced interaction between RecQL4 and RNF8 was observed upon the increased input of WRAP53β protein assessed by an in vitro Flag pull-down assay, followed by the western blotting analysis. C Dynamic recruitment of GFP-RNF8 to DSBs induced by UV micro-point laser in WRAP53β silenced U2OS cells relative to scrambled siRNA-transfected cells. D Dynamic recruitment of GFP-RecQL4 to DSBs in scrambled siRNA or WRAP53β siRNA-transfected U2OS cells.

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Immunoprecipitation, Transfection, In Vitro, Pull Down Assay, Purification, Recombinant, Western Blot

Journal: Journal of cell science

Article Title: The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability.

doi: 10.1242/jcs.02556

Figure Lengend Snippet: Fig. 3. Determination of the specificity of the RECQL4 antibodies by siRNA. (A) Western blot with anti-RECQL4 C-t antibody (upper panel) or anti-tubulin antibody (lower panel) using total cell extracts from HeLa cells transfected with 200 nM RECQL4-1 or control-1 siRNA. Asterisks indicate breakdown products. (B) RECQL4 knock-down leads to a loss of RECQL4 nuclear foci. Immunofluorescence analysis with anti-RECQL4 N-t antibody (red) on exponentially growing HeLa cells transfected with 200 nM RECQL4-1 or control-1 siRNA, DAPI- staining shows nuclear DNA (blue). Bar, ~10 m. (C) The mean number of RECQL4 foci per cell after siRNA treatment is displayed in histograms (standard error of the mean is indicated in the columns).

Article Snippet: For western blots, anti-RECQL4 N-t and anti RECQL4 C-t antibodies were used in a 1:2000 dilution, anti-tubulin antibody (Santa Cruz, TU02) in a 1:1000 dilution, anti-PARP1 antibody (Petrilli et al., 2004) in a 1:2000 dilution, anti-BLM antibody (Wu et al., 2000) in a 1:1500 dilution, anti-GRB2 antibody (BD Transduction, G10111) in a 1:2000 dilution, anti-FLAG antibody (Sigma, M2) in a 1:2000 dilution and anti-Rad51 antibody (Tan et al., 1999) in a dilution 1:20,000.

Techniques: Western Blot, Transfection, Control, Knockdown, Staining

Journal: Journal of cell science

Article Title: The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability.

doi: 10.1242/jcs.02556

Figure Lengend Snippet: Fig. 4. RECQL4 colocalizes with PML. HeLa cells were stained with anti-RECQL4 (red) and anti-PML (green) antibodies. Where red and green signals overlap, a yellow pattern is seen, indicating colocalization of RECQL4 and PML. DAPI-staining shows nuclear DNA. Bar, ~10 m. (Upper panel) Colocalization of RECQL4 and PML in untreated HeLa cells. (Lower panel) Colocalization of RECQL4 and PML after 10 M etoposide treatment of HeLa cells.

Article Snippet: In immunofluorescence experiments, anti-PML antibody (Santa Cruz, PG-M3) was used in a 1:50 dilution, anti-Rad51 antibody (abcam, ab1837) in a 1:50 dilution, anti-BrdU antibody (Pharmingen, 3D4) in a 1:50 dilution, anti-RECQL4 N-t antibody in a 1:200 dilution.

Techniques: Staining