Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Western blot immunoreactivity of the polyclonal anti-MPV17 antibody Ab 93374 ( left ) and the monoclonal anti- human MPV17 antibodies 6F5 and 5D2 on extracts of human and murine cells of different MPV17 genotypes. Left extracts from human U2OS cells, untransfected or transfected with the MPV17 expression clone SC118652 (origene), were probed with the antibody ab 93374 (abcam). Center untransfected U2OS cells with MPV17 +/+ endogenous genotype probed with the monoclonal antibodies 6F5 and 5D2. Right Murine embryo fibroblast (MEF) cells of MPV17 +/+ or MPV17 -/- genotype probed with the monoclonal antibodies 6F5 and 5D2. Indicated marker sizes were transferred from Ponceau stained marker bands run in parallel. Loading was controlled as indicated by reaction of the filters with an anti-ß-actin antibody. 10 μg of protein were separated on SDS gels and analysed by Western blot according to ( left panel ), and Kinkley et al. respectively

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Western Blot, Transfection, Expressing, Marker, Staining

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Ab93374 (Abcam) recognizes MPV17 specific structures in U2OS cells transiently transfected with an MPV17 expression construct in immunofluorescence analysis. Ab93374 signal ( green ) is largely missing in nontransfected cells. The immunoreactivity does colocalise with peroxisomal (anti-catalase, mouse polyclonal, Abcam ab88650) and mitochondrial (anti-complex IV mitochondrial subunit I, mouse monoclonal, Invitrogen 459600) markers ( red ). Immunofluorescence (IF) method was according to Pircher et al. using a MicroRadiance confocal scanning system (Bio-Rad) in combination with a Zeiss Axiophot microscope. Colocalisation analysis was performed using the Fiji software of Image J and is illustrated by white dots

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Transfection, Expressing, Construct, Immunofluorescence, Microscopy, Software

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: In U2OS cells, 5D2 antibody detects a punctate pattern colocalising with a peroxisomal (PMP70) but not with a mitochondrial (MitoTracker) marker. Top Rabbit anti PMP70 antibody ( decorated green ) was a gift from W. Just, Heidelberg. Secondary antibodies: Alexa Fluor 555 anti-mouse and Alexa Fluor 488 anti-rabbit. Bottom Mitotracker 7510 (Invitrogen) was used as recommended by the supplier. Secondary antibody: Alexa 488 anti-mouse. IF method and microscopy according to Kinkley et al. using a Zeiss LSM confocal microscope. Mathematical colocalisation analysis was performed using the Fiji software of Image J and is illustrated by white dots

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Marker, Microscopy, Software

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: 5D2 colocalisation with a marker of early endosomes (EEA1) and lysosomes (LAMP1) in U2OS cells. Top Partial colocalisation of 5D2 with EEA1 (rabbit anti-human). Bottom colocalisation of 5D2 with LAMP1 in U2OS cells. The IF method, secondary antibodies, and Image J analysis were used as described in the legend of Fig.

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Marker

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Colocalisation of 5D2 and LAMP1 mediated immunofluorescence in a U2OS single cell. IF and analysis of 5D2 and Colocalisation of 5D2 with LAMP1 in U2OS cells. The IF method, secondary antibodies, and Image J analysis were used as described in the legend of Fig. . In addition a merge of the two immunofluorescence pictures is depicted

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Immunofluorescence

Journal: Cell reports

Article Title: NEIL3 contributes to the Fanconi anemia/BRCA pathway by promoting the downstream double-strand break repair step.

doi: 10.1016/j.celrep.2022.111600

Figure Lengend Snippet: Figure 4. NEIL3 is required for DSB end resection (A–D) Representative images and statistical analysis to show (A, B) RPA2 foci and (C, D) BrdU foci formation 24 h after 100 ng/mL MMC treatment in WT and NEIL3/ HeLa cells. Scale bar, 10 mm. A total of 100 cells from three independent experiments were analyzed for each group. ***p < 0.001, ****p < 0.0001, two- tailed Student’s t test. (E) The schematic of in vitro DSB end resection assay in ER-AsiSI U2OS cells. See STAR Methods for details. (F) Quantification and statistical analysis of the ssDNA level after depletion of NEIL3, CtIP, and BRCA1, respectively, in ER-AsiSI U2OS cells. Data are shown as mean ± SEM compiled from three independent experiments. **p < 0.01, ***p < 0.001, two-tailed Student’s t test. (G) The efficiency of knockdown of individual proteins was examined by western blot.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 4-Hydroxytamoxifen Sigma-Aldrich Cat# H7904 Ethidium Bromide Sigma-Aldrich Cat# E8751 Doxycycline Selleck Chemicals Cat# S5159 Hoechst 33,342 Beyotime Technology Cat# C1029 Doxycycline Selleck Chemicals Cat# S5159 Benzonase Sigma-Aldrich Cat# E8263 MG-132 Selleck Chemicals Cat# S2619 Olaparib (AZD2281) Selleck Chemicals Cat# S1060 Critical commercial assays CellTiter-Glo Luminescent Viability Assay Promega Cat# G7570 Subcellular Protein Fractionation Kit Life Technologies Cat# 78840 CometAssay Single Cell Gel Electrophoresis Assay Trevigen Cat# 4250-050-K Hieff Clone Plus One Step Cloning Kit Yeasen Cat# 10911ES20 Immunoprecipitation (IP/CoIP) Kit Absin Cat# abs955 TIANamp Genomic DNA Kit TIANGEN Cat# DP304 PierceTM anti-HA magnetic beads Thermo Fisher Scientific Cat# 88836 PierceTM anti-c-Myc magnetic beads Thermo Fisher Scientific Cat# 88842 GFP-Trap magnetic beads Chromotek Cat# gtma-20 Experimental models: Cell lines 293T Cell Line ATCC N/A HeLa Cell Line ATCC N/A U2OS Cell Line ATCC N/A RPE1 Cell Line ATCC N/A U2OS-HA-ER-AsiSI Cell Line Gift from Dr. Gaëlle Legube N/A U2OS-FANCD2 / Cell Line Li et al., 2020 N/A HeLa-FANCA / Cell Line Li et al., 2020 N/A HeLa-NEIL3 / Cell Line Li et al., 2020 N/A HeLa-NEIL3-WT-Dox Cell line This paper N/A HeLa-NEIL3-del N term-Dox Cell line This paper N/A HeLa-NEIL3-del C term-Dox Cell line This paper N/A HeLa-NEIL3-del GRF2 -Dox Cell line This paper N/A HeLa-NEIL3-del GRF 1-2-Dox Cell line This paper N/A Oligonucleotides siRNA targeting sequence: negative Control: GGGTATCGACGATTACAAA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #1: GGGTGGATCATGTTATGGA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #2: GCTAATGGATCAGAACGTA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #50UTR: TGAGTTGCACAGCGGTATT This paper N/A siRNA targeting sequence: FANCA: TTTTTCCCTCTTGACCCTT Li et al., 2020 N/A siRNA targeting sequence: FANCD2: GGCTCAGGATTCTAATGTA Li et al., 2020 N/A siRNA targeting sequence: NBS1: CCAACTAAATTGCCAAGTA Sangon Biotech N/A (Continued on next page) e2 Cell Reports 41, 111600, November 8, 2022

Techniques: Two Tailed Test, In Vitro, Resection Assay, Knockdown, Western Blot

Journal: Cell reports

Article Title: NEIL3 contributes to the Fanconi anemia/BRCA pathway by promoting the downstream double-strand break repair step.

doi: 10.1016/j.celrep.2022.111600

Figure Lengend Snippet: Figure 6. NEIL3 acts upstream of CtIP and MRN complex to promote their recruitment to DSBs (A) Western blots to validate the expression of GFP-tagged constructs of NEIL3, CtIP, and NBS1, respectively. (B) Representative time-lapse view to show the recruitment of indicated proteins to laser irradiation damaged sites in U2OS cells. Scale bar, 5 mm. (C) Normalized fluorescent intensity curve of indicated proteins at DNA damage sites. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 4-Hydroxytamoxifen Sigma-Aldrich Cat# H7904 Ethidium Bromide Sigma-Aldrich Cat# E8751 Doxycycline Selleck Chemicals Cat# S5159 Hoechst 33,342 Beyotime Technology Cat# C1029 Doxycycline Selleck Chemicals Cat# S5159 Benzonase Sigma-Aldrich Cat# E8263 MG-132 Selleck Chemicals Cat# S2619 Olaparib (AZD2281) Selleck Chemicals Cat# S1060 Critical commercial assays CellTiter-Glo Luminescent Viability Assay Promega Cat# G7570 Subcellular Protein Fractionation Kit Life Technologies Cat# 78840 CometAssay Single Cell Gel Electrophoresis Assay Trevigen Cat# 4250-050-K Hieff Clone Plus One Step Cloning Kit Yeasen Cat# 10911ES20 Immunoprecipitation (IP/CoIP) Kit Absin Cat# abs955 TIANamp Genomic DNA Kit TIANGEN Cat# DP304 PierceTM anti-HA magnetic beads Thermo Fisher Scientific Cat# 88836 PierceTM anti-c-Myc magnetic beads Thermo Fisher Scientific Cat# 88842 GFP-Trap magnetic beads Chromotek Cat# gtma-20 Experimental models: Cell lines 293T Cell Line ATCC N/A HeLa Cell Line ATCC N/A U2OS Cell Line ATCC N/A RPE1 Cell Line ATCC N/A U2OS-HA-ER-AsiSI Cell Line Gift from Dr. Gaëlle Legube N/A U2OS-FANCD2 / Cell Line Li et al., 2020 N/A HeLa-FANCA / Cell Line Li et al., 2020 N/A HeLa-NEIL3 / Cell Line Li et al., 2020 N/A HeLa-NEIL3-WT-Dox Cell line This paper N/A HeLa-NEIL3-del N term-Dox Cell line This paper N/A HeLa-NEIL3-del C term-Dox Cell line This paper N/A HeLa-NEIL3-del GRF2 -Dox Cell line This paper N/A HeLa-NEIL3-del GRF 1-2-Dox Cell line This paper N/A Oligonucleotides siRNA targeting sequence: negative Control: GGGTATCGACGATTACAAA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #1: GGGTGGATCATGTTATGGA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #2: GCTAATGGATCAGAACGTA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #50UTR: TGAGTTGCACAGCGGTATT This paper N/A siRNA targeting sequence: FANCA: TTTTTCCCTCTTGACCCTT Li et al., 2020 N/A siRNA targeting sequence: FANCD2: GGCTCAGGATTCTAATGTA Li et al., 2020 N/A siRNA targeting sequence: NBS1: CCAACTAAATTGCCAAGTA Sangon Biotech N/A (Continued on next page) e2 Cell Reports 41, 111600, November 8, 2022

Techniques: Western Blot, Expressing, Construct, Irradiation