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

Journal: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/elife.85601

Figure Lengend Snippet: Figure 1. Dynamics of caveolae at the rear of migrating RPE1 cells. (A) RPE1 cells were fixed and stained with anti-Cav1 antibodies. The rear localisation index (fold enrichment at the rear) was determined by measuring the Cav1 intensity at the cell front and the cell rear (n = 36 cells). Scale bar 10 µm. (B) Pearson’s correlation analysis of fixed RPE1 cells stably expressing cavin1-A2E stained with anti-Cav1 antibodies. (C) Time-lapse images of RPE1 cells stably transfected with Cavin1-A2E migrating on a glass coverslip. The front-rear distribution of Cavin1-A2E in each frame of the movie was measured using line scans. The mean localisation was plotted. Error bars indicate SEM. Scale bar 10 µm. (D) Time-lapse images of RPE1 cells stably transfected with Cavin3-miniSOG-mCherry migrating on a glass coverslip. The front-rear distribution of Cavin3 in each frame of the movie was measured using line scans. The mean localisation was plotted. Error bars indicate SEM. Scale bar 10µm. (E) Time-lapse images of RPE1 cells stably transfected with cavin3- miniSOG-mCherry migrating on in a collagen matrix. Scale bar 5 µm. (F) Time-lapse images of RPE1 cells stably transfected with cavin3-miniSOG- mCherry showing a sudden rear retraction. Scale bar 10µm. (G) Temporal relationship between front protrusion, rear retraction, and cell area of the cell shown in (F). (G) Temporal relationship between front protrusion, rear retraction, and aspect ratio (cell shape) of the cell shown in (F).

Article Snippet: RPE1 cell culture and transfection Human (h)TERT- RPE1 (CRL- 4000) cells were purchased from ATCC and tested for mycoplasma on a regular basis using a PCR- based mycoplasma test kit.

Techniques: Staining, Stable Transfection, Expressing, Transfection

Journal: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/elife.85601

Figure Lengend Snippet: Figure 2. Electron tomography of caveolae at the RPE1 cell rear. (A) Electron tomography of caveolae and caveolar networks at the rear of RPE1 cells stably transfected with Cavin3-miniSOG-mCherry. Two slices of the tomogram are shown. Note that individual caveolae are interconnected. (B) 3D surface model of the tomogram shown in (A), showing interconnected clusters, or rosettes, of caveolae. Scale bar in (A) and (B) is 100 nm.

Article Snippet: RPE1 cell culture and transfection Human (h)TERT- RPE1 (CRL- 4000) cells were purchased from ATCC and tested for mycoplasma on a regular basis using a PCR- based mycoplasma test kit.

Techniques: Tomography, Stable Transfection, Transfection

Journal: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/elife.85601

Figure Lengend Snippet: Figure 3. Proximity labelling of caveolae in RPE1 cells stably expressing Cav1-APEX2-EGFP. (A) Western blot of stable RPE1 cell lines expressing either Cav1-A2E or NES-A2E fusion proteins. The membrane was probed with anti-Cav1 antibodies. (B) Confocal fluorescence microscopy of Cav1-A2E RPE1 cells stained with anti-PTRF/cavin1 antibodies. Boxed regions are magnified in the bottom panel. Nuclei were counterstained with DAPI. Scale bar 10 µm. (C) Transmitted light image of Cav1-A2E RPE1 cells after APEX2 labelling and plastic embedding (left) and correlative TEM image of the same cell (right). (D) Representative TEM micrographs of serial TEM sections of the cell shown in (C) (boxed regions). Note the specific labelling and abundance of caveolae at the cell rear. (E) Cav1-A2E-expressing cells were incubated in the absence or presence of biotin phenol and/or hydrogen peroxide. Cell lysates were separated by SDS-PAGE, blotted onto PVDF membrane, and probed with streptavidin-HRP. (F) Confocal microscopy of Cav1-A2E RPE1 cells after proximity labelling with APEX2. Cells were fixed and stained with fluorescent streptavidin (Alexa Fluor 555) to visualise biotinylated proteins. Nuclei were counterstained with DAPI. Scale bar 10 µm.

Article Snippet: RPE1 cell culture and transfection Human (h)TERT- RPE1 (CRL- 4000) cells were purchased from ATCC and tested for mycoplasma on a regular basis using a PCR- based mycoplasma test kit.

Techniques: Stable Transfection, Expressing, Western Blot, Membrane, Fluorescence, Microscopy, Staining, Incubation, SDS Page, Confocal Microscopy

Journal: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/elife.85601

Figure Lengend Snippet: Figure 5. Proximity ligation assays (PLA) reveal dynamic interactions between caveolae and the cortical actin cytoskeleton during changes in membrane tension. (A–D) PLA in the Cav1-A2E RPE1 cell line using anti-GFP and anti-PTRF/cavin1 (A, B), anti-FLNA (C), or anti-CTTN (D) antibodies as indicated. (E, F) PLA in RPE1 cells transfected with EHD2-A2E using anti-GFP and anti-Cav1 antibodies. (G) Quantification of rear-localised PLA signals based on data shown in panels (A–F). (H–K) Effect of ROCK inhibition on PLA in the Cav1-A2E RPE1 cell line using the indicated combinations of antibodies. Cells were incubated for 5 min in hypo-osmotic medium containing 10 µM Y-27632 or DMSO (control) and then transferred to isotonic medium for 30 min in the absence (DMSO) or presence of Y-27632. The graphs in (B, C, D, F, J, K) show the quantifications of the PLA signals per cell. 30 cells from three independent experiments were quantified for each treatment. The number of PLA dots per cell is presented with the mean values ± SD. The bottom panels in (A, E, H, I) show the PLA signal masks used for counting. Statistical significance was calculated using a Wilcoxon test. ns = p>0.05, ***p≤0.001. All scale bars 20 µm.

Article Snippet: RPE1 cell culture and transfection Human (h)TERT- RPE1 (CRL- 4000) cells were purchased from ATCC and tested for mycoplasma on a regular basis using a PCR- based mycoplasma test kit.

Techniques: Ligation, Membrane, Transfection, Inhibition, Incubation, Control

Journal: eLife

Article Title: Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

doi: 10.7554/elife.85601

Figure Lengend Snippet: Figure 6. Evidence for a functional connection between caveolae and ARHGAP29. (A) RPE1 cells were transfected with siRNAs against ARHGAP29, caveolin-1, or non-targeting siRNAs and analysed by western blotting 48 hr post-transfection. (B) Quantification of western blot analysis shown in (A). Ratios normalised to the GAPDH loading control are displayed relative to the intensity of the control siRNA transfection for each protein indicated. Data represent the mean ± SD of 3–4 independent experiments. Statistical significance was calculated using an unpaired t-test. ns = p>0.05, *p≤0.05,

Article Snippet: RPE1 cell culture and transfection Human (h)TERT- RPE1 (CRL- 4000) cells were purchased from ATCC and tested for mycoplasma on a regular basis using a PCR- based mycoplasma test kit.

Techniques: Functional Assay, Transfection, Western Blot, Control

Journal: Scientific reports

Article Title: FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress.

doi: 10.1038/s41598-021-93708-5

Figure Lengend Snippet: Figure 1. Schematic of RPE cell interactions with their underlying ECM. (a) The RPE forms a monolayer on Bruch’s membrane and plays a crucial role in maintaining photoreceptors. Bruch’s membrane separates the RPE from its blood supply, the choriocapillaris. RPE cells adhere to, and interact with, ligands within the basement membrane of Bruch’s membrane (BrM), including FN and LA, through various integrin heterodimer receptors expressed on their surface. Bruch’s membrane comprises five separate layers: the RPE basement membrane; the inner collagenous layer; an elastin core; the outer collagenous layer; and the endothelial basement membrane layer. The complement regulator FHL-1 is found anchored to heparan sulphate and dermatan sulphate glycosaminoglycan (GAG) chains found in collagenous and basement membrane layers of Bruch’s membrane. (b) Both FH and its truncated variant FHL-1 are comprised of complement control protein (CCP) domains: FH has twenty domains, while FHL-1 has seven CCPs identical to FH but with a unique C-terminal tail. Both proteins also share an RGD integrin-binding motif in CCP4.

Article Snippet: The human immortalized RPE cell line hTERT RPE-1 (ATCC CRL-400) was purchased from ATCC (LGC Standards, UK).

Techniques: Membrane, Variant Assay, Control, Binding Assay

Journal: Scientific reports

Article Title: FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress.

doi: 10.1038/s41598-021-93708-5

Figure Lengend Snippet: Figure 3. RPE cell interactions with FHL-1 are mediated through the α5β1 integrin. Cultured primary RPE cells (10,000 cells per well of a 96-well plate) were incubated on an FHL-1 matrix (a) in the presence of competing antibodies against specific integrin subunits (all 10 µg/ml), including: (b) anti-α5; (c) anti-β1; (d) anti-αV; (e) anti-αVβ3; and (f) anti-αVβ5. (g) The percentage of cell spreading on FHL-1 in the presence of each inhibiting antibody was calculated. The inhibitory effects of both anti-β1 and anti-α5 on primary RPE cell spreading on FHL-1 were shown to be dose dependent in (h) and (i) respectively. Data are the average results of three independent experiments and each performed in triplicate. Images in (a-f) are representative of 3 independent experiments. Data in (g) represent n = 3 ± s.e.m. Statistical analysis was performed by paired One- Way ANOVA with Tukey post hoc analysis, where *P < 0.05 and ***P < 0.001. Data in (h-i) are n = 3 ± s.e.m. Scale bar represents 100 μm.

Article Snippet: The human immortalized RPE cell line hTERT RPE-1 (ATCC CRL-400) was purchased from ATCC (LGC Standards, UK).

Techniques: Cell Culture, Incubation

Journal: Scientific reports

Article Title: FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress.

doi: 10.1038/s41598-021-93708-5

Figure Lengend Snippet: Figure 4. RPE cell α5β1 integrin recognises the RGD binding motif in FHL-1. (a) Schematic showing the design of both the RGD cyclic peptide and the scrambled peptide control, as well as the location of the aspartic acid mutation to an alanine residue for the creation of the RGD null FHL-1 protein. (b) The spreading of primary RPE cells to immobilised FHL-1 was inhibited with increasing concentrations of the cyclic RGD peptide: no inhibition was observed under the same conditions with the control scrambled peptide. Data comprises n = 3 ± s.e.m. Statistical analysis was performed using Student’s t test at each peptide conc., where **P < 0.01, and ***P < 0.001. The binding of the α5β1 integrin to the RGD binding domain in FHL-1 is tested by incubation of primary RPE cells with (c) fibronectin, (d) FHL-1, (e) FHL-1 RGD null, or (f) BSA. (g) Percentage of spread cells were calculated and compared to the positive control, fibronectin. Data are the average results of the three independent experiments and each performed in triplicate. Images in (c-f) are representative of 3 independent experiments. Data in (g) represent n = 3 ± s.e.m. Scale bar represents 100 μm.

Article Snippet: The human immortalized RPE cell line hTERT RPE-1 (ATCC CRL-400) was purchased from ATCC (LGC Standards, UK).

Techniques: Binding Assay, Control, Mutagenesis, Residue, Inhibition, Incubation, Positive Control