Reddot2

Image cytometry with an NC-3000 Image Cytometer (ChemoMetec, Allerod, Denmark) was used for cellular and mitochondrial phenotyping. A minimum number of 5000 events were analyzed in each experiment. The following assays were executed according to the manufacturer’s recommendations: cell count (acridine orange and DAPI (ChemoMetec)), mitochondrial membrane potential (JC-1 and DAPI (ChemoMetec)), thiol redox status (VitaBright-48, Propidium iodide, and acridine orange (ChemoMetec)). Mitochondrial superoxide levels (MitoSOX™ (Thermo Scientific) and Hoechst-33342 (ChemoMetec)) were measured as described earlier [43 (link)]. To measure mitochondrial mass, 100 nM fluorescent dye MitoTracker™ Green FM (Invitrogen, Carlsbad, CA, USA) diluted in Hank’s balanced salt solution (HBSS) (Invitrogen) was added to cells for incubation for 30 min at 37 °C. After removing excess dye, the cells were collected and a 1:1000 dilution of RedDot2 (Biotium, Fremont, CA, USA) (concentration not specified) was added to each sample before measurement. The NucleoView NC-3000 software (ChemoMetec) was used for data analysis.

Mice were anesthetized and transcardially perfused with 4% PFA (w/v) in PBS (pH 7.4). Brains were removed, post-fixed for 2 hr in 4% PFA, cryo-protected in 30% (w/v) sucrose at 4°C and embedded in Tissue-Tek medium (Sakura Finetek Europe, The Netherlands). Free-floating cryosections (40 µm) were washed three times in Tris-Buffered Saline (TBS), permeabilized in 0.3% (v/v) Triton-X-100 in TBS for 5 min and incubated with blocking solution (10% (v/v) normal serum, 1% (w/v) BSA, 0.3% Triton-X-100 in TBS) for 2 hr. Sections were incubated with primary antibodies overnight at 4°C. After three washes in 0.025% Triton-X-100 in TBS sections were incubated with corresponding secondary antibodies. Sections were counterstained with Hoechst 33258 (H-3569, Molecular Probes, Grand Island, NY) for 5 min or with RedDot2 (40061, Biotium, Fremont, CA) for 20 min. After three washes in TBS sections were mounted with Roti-Mount FluorCare mounting medium (Carl Roth, Germany). Images were acquired using a LSM 510/Axiovert200M microscope (Carl Zeiss, Germany).
For cresyl violet staining free-floating brain sections (40 µm) were mounted on glass slides and incubated in cresyl violet (0.1 M sodium acetate, 2% acetic acid, 0.02 M cresyl violet acetate, Sigma, St. Louis, MO, in distilled water, pH 3.5).

Nontransformed rat intestinal epithelial IEC-6 cells (ATCC CRL-1592) were cultured as described before (14 (link)). Experimental procedures were described in Text S1 and Fig. S1A. At the end of the treatments, in-cell Western (ICW) procedure and immunofluorescence analysis were performed to analyze DNA damage via phosphorylation of the histone H2AX.
The ICW procedure was performed as previously described (53 (link)). The cells were fixed, permeabilized, blocked, and then incubated overnight (ON) with rabbit monoclonal anti-γH2AX 1/200 (20E3; Cell Signaling, Saint-Quentin en Yvelines, France). An infrared fluorescent secondary antibody (IRDye 800CW; Rockland) (1/500) was applied simultaneously with RedDot2 (1/500) (Biotium, Interchim, Montluçon, France) for DNA labeling. The DNA and γH2AX were visualized using an Odyssey infrared imaging scanner (LI-COR Science Tec, Les Ulis, France). All experiments were carried out in triplicate.

Anti 53BP1 (Novus Biological) from rabbit is diluted 1/3000 in PBS containing 3% bovine serum albumin (BSA), and anti γH2AX (Merck/Millipore) from mouse is diluted 1/3000. For In-Cell Western, anti γH2AX (Cell Signaling technology) from rabbit was diluted 1/200 in PBS containing 2% fetal bovine serum (FBS) and 0.2% Triton X-100 (PST buffer).
Alexa fluor 546 from rabbit and 488 (Invitrogen) from mouse were diluted 1/800 in PBS. Goat anti-rabbit antibody coupled with 770-nm fluorophore (diluted 1/1000 in PST buffer) and RedDot2 (diluted 1/1000 in PST buffer) were purchased from Biotium.
Anti β-catenin (mouse monoclonal, Santa Cruz technologies), Phalloidin-TRITC (Sigma-Aldrich) and Chicken anti-mouse Alexa 488 (Fisher scientific) were diluted 1/100, 1/10,000, and 1/2000, respectively, in PBS containing 3% BSA and 0.1% Triton X-100.
APC protein was detected by a mouse monoclonal antibody (Ab1, clone FE9) against the N-terminal end (Calbiochem) and lamins A/C with a monoclonal antibody (clone 4C11, Sigma-Aldrich). Donkey anti-mouse IgG DyLight 800 conjugate (Thermoscientific) was used as secondary antibody. Anti APC, anti lamin and secondary antibody were diluted in TBS/BSA3%/0.1% Tween (1/100; 1/10,000, and 1/20,000, respectively).

Pharmacological inhibitors were prepared in either DMSO or 20 % acetonitrile/water according to the manufacturer’s recommendations and used at the indicated concentrations. EIPA, CPZ, Gen, and Rot were purchased from Sigma Aldrich. The Rac1 inhibitor W56 was purchased from Tocris Bioscience.
Specific antibodies including mouse anti-beta actin (ab8226), rabbit anti-EEA1 antibody (ab2900), Anti-LAMP1 [H4A3], anti-beta actin antibody [AC-15], anti-beta tubulin antibody, anti-neuron specific beta III tubulin were purchased from Abcam. Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, streptavidin Alexa Fluor 633 conjugate, streptavidin Alexa Fluor 488 conjugate, Alexa Fluor 488 donkey anti-sheep, Alexa Fluor 488 donkey anti-rabbit, SYTOX Red dead cell stain, FM® 1-43FX fixable analogue of FM® 1–43 membrane stain were purchased from Invitrogen Life Technologies. Donkey anti-sheep/goat IgG HRP conjugate and goat anti-mouse IgM + IgG + IgA (H + L) HRP conjugates were purchased from Millipore.
Sheep anti-SOD1 was purchased from Thermo Fisher Scientific. Mouse monoclonal anti-human TARDBP antibody (clone k1B8) was purchased from Abnova. Anti-BiP/GRP78 was purchased from BD Transduction Laboratories. FITC-conjugated sheep anti-mouse was purchased from Silenus. RedDot 2 was obtained from Biotium. Goat Anti-Rabbit IgG (H + L)-HRP Conjugate was obtained from Bio-Rad.

Immunofluorescence staining was performed as described previously^{65 (link)}. In brief, pseudoblastocysts (96 or 120 hpi) or ESCs were fixed for 20 min at room temperature with 4% paraformaldehyde in PBS, washed with PBS containing 0.05% Tween 20 (PBST), and permeabilized for 30 min at room temperature with 0.4% Triton X-100 in PBS. They were then washed with PBST before incubation first for 1 h at room temperature with 1% BSA in PBST and then overnight at 4 °C with rabbit anti-CDX2 (1:1000 dilution, Abcam ab76541), mouse anti-OCT4 (1:500 for embryos and 1:250 for ESCs, Santa Cruz sc-5279), rabbit anti-NANOG (1:200, Abcam ab80892), mouse anti-GATA4 (1:200, Santa Cruz sc-25310; 1:1000, Santa Cruz sc-25310AF548), or mouse anti-DAB2 (1:1000, Santa Cruz sc-136964AF488) in the same solution. They were again washed with PBST, incubated for 1 h at room temperature with appropriate Alexa Fluor 488– or Alexa Fluor 568–conjugated secondary antibodies (Molecular Probes–Invitrogen), washed with PBST, and mounted in SlowFade Gold antifade reagent (Molecular Probes–Invitrogen) containing Hoechst 33342 (5 μg/ml) or RedDot2 (1:200, Biotium). Fluorescence images were acquired at multiple 1-μm intervals in the z-axis with the use of a confocal microscope (CV1000, Yokogawa).

Quantification of DNA DSBs was performed using the In Cell Western assay as described [25 ]. Caco-2 cells were dispensed (1 × 10⁵ cells/well) in a black 96 well plate (Greiner Bio-One) and incubated at 37 °C in 5% CO₂ atmosphere. After 24 h, Caco-2 cells were infected at MOI 50 with E. coli strains. After 4 h of infection, the cells were washed three times with PBS and incubated for 3 h in cell culture medium supplemented with 200 μg/ml gentamicin. Cells were fixed (4% paraformaldehyde), permeabilized, blocked and then incubated overnight with rabbit monoclonal anti-γ-H2AX (BioLabs) at 1/200 dilution. Secondary antibody IRDye™800CW goat anti-rabbit (Biotium, Wisconsin, United States) was applied simultaneously with 1/500 dilution of RedDot™2 (Biotium) for DNA labeling. The DNA and γ-H2AX were visualized using Odyssey® infrared imaging scanner (LI-COR model 9120, Québec, Canada) with red denoting RedDot™2 and green for IRDye™800CW goat anti-rabbit. Images were processed using Image Studio Ver3.1 software.