Journal: bioRxiv

Article Title: Compromised nuclear envelope integrity drives tumor cell invasion

doi: 10.1101/2020.05.22.110122

Figure Lengend Snippet: (A) RPE1 cells stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry were confined at the indicated heights and images were acquired while cells were under confinement (after 2 h). Bar, 5 μm. (B, C) Quantification of DNA damage levels during confinement as assessed by the number of 53BP1 foci in cells displaying or not NE rupture. Data represents the mean ± SEM of 3 independent experiments where 20 cells per experiment and per condition were analyzed. Red vertical line indicates a single time-point, which is represented in “B”. (D, E) Frequency of foci appearance and foci life-time (foci duration) for RPE1 cells confined at 2 μm (with or without NE rupture) or treated with etoposide (25 μM). Data represents the mean ± SD of 3 independent experiments where 20 cells per condition were analyzed. (F) A variety of TREX1-deficient or -proficient RPE1 cells (all stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry) were confined at 2 μm height and DNA damage levels were assessed after 2h under confinement by the number of 53BP1 foci in cells displaying or not NE rupture. (G) Same as in “F” but with DCIS cells. (F, G) Data represents the mean ± SEM of 3 independent experiments where 20 cells were analyzed per condition per experiment. Western blots show TREX1 depletion in RPE1 and DCIS cells 48h postknockdown; tubulin is the loading control. Bar, 10 μm. P values were calculated by unpaired Student’s t -test, ***P < 0.0001; **P < 0.005; ns = not significant.

Article Snippet: The following pharmacological inhibitors and chemical compounds were used: Etoposide (DNA damaging agent, topoisomerase-II inhibitor; Cell Signaling, #2200), Doxorubicin (DNA damaging agent, topoisomerase-II inhibitor; Cell Signaling, #5927), GM6001 (pan-MMP inhibitor); Millipore, # CC1010), KU-55933 (ATM kinase inhibitor; Abcam, #120637).

Techniques: Stable Transfection, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Compromised nuclear envelope integrity drives tumor cell invasion

doi: 10.1101/2020.05.22.110122

Figure Lengend Snippet: (A) RPE1 cells stably expressing catalytically inactive cGAS-EGFP were confined at the indicated heights and images were acquired immediately after (and while cells were under confinement). Bar, 15 μm. (B) Quantification of NE rupture events as assessed by cGAS perinuclear localization. Data represents the mean ± SD of 3 independent experiments where 50 cells per experiment per height were analyzed. (C) MCF10A cells stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry were transiently depleted for TREX1 with siRNA and 48 h later cells were confined at 2 μm. (D) Quantification of DNA damage levels during 2 μm confinement as assessed by the number of 53BP1 foci in siControl and siTREX1 cells displaying or not NE rupture. Western blot shows TREX1 depletion 48 h post-knockdown; tubulin is the loading control. Bar, 10 μm. Data represents the mean of 3 independent experiments where 20 cells per experiment per condition were analyzed. (E) RPE1 cells stably expressing catalytically inactive cGAS-EGFP were confined at 2 μm for 2 h; subsequently, the confinement lid was removed, cells were harvested, replated for 30 minutes and fixed for immunostaining with the DNA damage markers RIF1 and γH2AX (red) and DAPI (blue). Arrowheads point to cells with NE rupture, which are also positive for the DNA damage markers. Bar, 20 μm. (F) Quantification of DNA damage foci at the indicated conditions (DMSO, etoposide 25 μM and 2 μm confinement). Images are representative of 2 independent experiments where 30 cells per experiment per condition were analyzed. Bars represent the mean ± SD. (G) RPE1 cells stably expressing 53BP1-EGFP were confined at 2 μm and imaged under spinning disc microscopy. White arrowhead points to a NE bleb while the yellow arrowhead points to a bleb bursting event that is followed by the appearance of DNA damage foci. (H) Graphs showing the absolute number of 53BP1 foci and the events of appearance of new 53BP1 foci (red asterisks) following a bleb bursting event (assessed by an increase in the cytosolic intensity of the probe 53BP1-EGFP, which leaks out of the nucleus upon NE rupture). Blue arrow indicates the instant of NE rupture/bleb bursting. (I) Diagrams illustrating the Cas9/gRNA targeting sequence in the TREX1 gene and the primers used for the sequencing of the TREX1 KO clones generated by CRISPR technology for RPE1 and DCIS cells. (J) Western blots of WT and TREX1 KO clones; tubulin is a loading control. (K) RPE1 cells stably expressing 53BP1-EGFP were transiently depleted for TREX1 using siRNA and treated with etoposide (25 μM) for 2 h. Subsequently cells fixed and stained for endogenous TREX1. Bar, 25 μm. (L) Frequency distribution of the number of nuclear blebs in RPE1 TREX1 KO clones and in parental RPE1 cells. Data represents 3 independent experiments where 60 cells per experiment were analyzed. (M) RPE1 cells transiently transfected with TREX1-EGFP WT were confined at 2 μm using a pressure-controlled dynamic confiner. Images represent a single plane Z slice through the middle section of the nucleus. Bars, 5 μm and 1 μm (magnification). Images are representative of 2 independent experiments.

Article Snippet: The following pharmacological inhibitors and chemical compounds were used: Etoposide (DNA damaging agent, topoisomerase-II inhibitor; Cell Signaling, #2200), Doxorubicin (DNA damaging agent, topoisomerase-II inhibitor; Cell Signaling, #5927), GM6001 (pan-MMP inhibitor); Millipore, # CC1010), KU-55933 (ATM kinase inhibitor; Abcam, #120637).

Techniques: Stable Transfection, Expressing, Western Blot, Knockdown, Control, Immunostaining, Microscopy, Sequencing, Clone Assay, Generated, CRISPR, Staining, Transfection

Journal: bioRxiv

Article Title: Compromised nuclear envelope integrity drives tumor cell invasion

doi: 10.1101/2020.05.22.110122

Figure Lengend Snippet: (A) RPE1, MCF10A and DCIS cells were treated with doxorubicin (40 nM), etoposide (25 μM) or DMSO (vehicle) for the indicated lengths of time. Cells were then fixed and processed for β-gal staining. The percentage of β-gal-positive cells was plotted. Graph: data represents mean ± SD of 3 independent experiments where 200 cells were scored per condition per experiment. (B) Frequency distribution (percentage) of the designated categories for cells under the indicated confinement heights. Data represents 3 independent experiments where 37 non-confined cells, 40 confined cells with non-ruptured nuclei and 60 confined cells with ruptured nuclei were scored per condition per experiment. (C) RPE1 cells were confined at 2 μm in the presence of propidium iodide and imaged overnight. Graph: data represents the mean ± SD of 2 independent experiments where 5 random fields (100 cells scored per field) were analyzed per time-point per experiment. Bar, 50μm. (D) Epifluorescence and phase images of RPE1 cells stably expressing catalytically inactive cGAS-EGFP harvested from 2 μm confinement and replated for cell cycle duration measurement over a 72 h period. Bar, 25 μm. Inset shows a cell with ruptured NE, as evidenced by cGAS perinuclear accumulation. (E) MCF10A cells or (F) DCIS cells were confined for 12 h at 10 or 2 μm, harvested from confinement and replated for 72 h before fixation for immunostaining with heterochromatin foci (HP1γ) or lysis for western blot analysis of lamin B1 and p21. GAPDH is the loading control; western blot images are representative of 2 independent experiments. Graph: box and whisker plot showing the median value and 10-90 percentiles of the number of HP1γ foci per nucleus. Data represents 3 independent experiments where 100 cells per condition per experiment were analyzed. Bars, 10 μm. (G) RPE1 cells were confined for 12 h at 4 or 2 μm; cells were then harvested from confinement and replated for 72 h before lysis for western blot analysis of lamin B1 and p21. Tubulin is the loading control; western blot images are representative of 2 independent experiments. (H) Western blot analysis of whole cell extracts of human fibroblasts, RPE1 cells and HeLa cells. GAPDH is the loading control. P values were calculated by unpaired Student’s t -test, ***P < 0.0001; *P < 0.05; ns = not significant.

Article Snippet: The following pharmacological inhibitors and chemical compounds were used: Etoposide (DNA damaging agent, topoisomerase-II inhibitor; Cell Signaling, #2200), Doxorubicin (DNA damaging agent, topoisomerase-II inhibitor; Cell Signaling, #5927), GM6001 (pan-MMP inhibitor); Millipore, # CC1010), KU-55933 (ATM kinase inhibitor; Abcam, #120637).

Techniques: Staining, Stable Transfection, Expressing, Immunostaining, Lysis, Western Blot, Control, Whisker Assay

Journal: bioRxiv

Article Title: Genetic factors and age are the strongest predictors of humoral immune responses to common pathogens and vaccines

doi: 10.1101/254706

Figure Lengend Snippet: Serum samples from the 1,000 age‐ and sex-stratified healthy individuals of the Milieu Intérieur cohort were used for measuring total antibody levels (IgA, IgM, IgG and IgE), as well as for qualitative (serostatus) and quantitative (IgG levels) assessment of IgG responses against cytomegalovirus, Epstein-Barr virus (anti-EBNA, anti-VCA, anti-EA), herpes simplex virus 1 & 2, varicella zoster virus, Helicobacter pylori , Toxoplasma gondii , influenza A virus, measles, mumps, rubella, and hepatitis B virus (anti-HBs and anti-HBc), using clinical-grade serological assays.

Article Snippet: Anti-HSV1 and anti-HSV2 IgGs were measured using the BioPlex 2200 HSV-1 & HSV-2 IgG kit on the BioPlex 2200 analyzer (Bio-Rad).

Techniques: