Journal: bioRxiv

Article Title: Organ Chips with integrated multifunctional sensors enable continuous metabolic monitoring at controlled oxygen levels

doi: 10.1101/2024.08.08.606660

Figure Lengend Snippet: A. Percentage change of impedance in Gut siChip with time during EGTA treatment and after it was removed. B. Continuous measurement of percentage change in oxygen level during EGTA treatment and post recovery. C. Change in the Gut siChip oxygen level (black line) and impedance (blue line) in response to oligomycin (+OM) or its vehicle (-OM) exposure and after they were removed. D. Immunofluorescence images (top) of siChip intestinal epithelium 48 hours after treatment with OM compared to drug carrier stained with DAPI (nuclei, blue) and H2AX (DNA damage, magenta). Image-based quantitative analysis of percentage change in epithelial cells DNA damage with vehicle and OM treatment (bottom). Scale bar 100 μm. E. Measurements of pH changes in outflows of epithelium (black line) and endothelium (blue line) lumens in response to Lactococcus Lactis ( L. lactis ) bacteria co-culture. F. Immunofluorescent microscopy images of the gut epithelium stained with DAPI (nuclei, blue) and F-actin (magenta) markers 96 hours after inoculation with L. lactis bacteria. Scale bar 100 μm. Data represent the mean ± s.e.m.; n=5 ( A ), 3 ( B ), 3-6 ( C ), and 3-6 ( E ) experimental chip replicate. * P< 0.05, ** P< 0.01, *** P< 0.001. Presented percentage and fold change values are normalized to pre-treatment levels. Red arrows indicate the end of EGTA or OM treatment in A,B, and C.

Article Snippet: At the endpoint of experiments, siChips were removed from the mPods, washed two times with DPBS, fixed with 4% paraformaldehyde for 15-20 minutes at room temperature, and incubated overnight in 1:100 anti-H2AX antibody (Novus Bio NB100-2280) at 4°C in 1% BSA in PBS and 0.01% Triton-X 100.

Techniques: Immunofluorescence, Staining, Bacteria, Co-Culture Assay, Microscopy

Journal: bioRxiv

Article Title: Organ Chips with integrated multifunctional sensors enable continuous metabolic monitoring at controlled oxygen levels

doi: 10.1101/2024.08.08.606660

Figure Lengend Snippet: Dynamic fold change in oxygen level ( A ) and percentage change in impedance ( B ) of Liver siChip throughout exposure to EGTA and after its removal. C. Quantitative analysis of epithelial cell size before and after 3 hours of EGTA treatment compared to control chips with no EGTA. D. Immunofluorescent microscopic images (left) and quantitative analysis (right) of the DNA damage (H2AX + cells, magenta) in epithelium after exposure to OM compared to drug carrier. Nuclei stained with DAPI (blue). Scale bar 100 μm. E. Dynamic changes in oxygen level (black line) and barrier impedance (blue line) after OM treatment and its removal compared to drug vehicle treated (-OM) siChips. Presented percentage and fold change values are normalized to pre-treatment levels. F. Representative images of ATP/ADP fluorescent signal intensity (left) and image-based quantitative analysis (right) before and after 5 hours of exposure to OM (+OM) or drug vehicle (-OM). Scale bar 100 μm. Data represent the mean ± s.e.m.; n=5 ( A ), 5 ( B ), 10 ( C ), 6-15 ( E ) and 6 ( F ) experimental chip replicate * P< 0.05, ** P< 0.01, *** P< 0.001, **** P< 0.0001. Red arrow indicates the end of EGTA or OM treatment in A, B , and E.

Article Snippet: At the endpoint of experiments, siChips were removed from the mPods, washed two times with DPBS, fixed with 4% paraformaldehyde for 15-20 minutes at room temperature, and incubated overnight in 1:100 anti-H2AX antibody (Novus Bio NB100-2280) at 4°C in 1% BSA in PBS and 0.01% Triton-X 100.

Techniques: Control, Staining

Journal: Nucleic acids research

Article Title: LINE-1 mRNA 3' end dynamics shape its biology and retrotransposition potential.

doi: 10.1093/nar/gkad1251

Figure Lengend Snippet: Figure 4. The effects of XRN1 knock-out on L1 cytoplasmic bodies and on L1-ORF2p . ( A ) Confocal microscopy z-stacks of 293T wild-type cells transfected with the L1 -ORF1 -EGFP expressing construct. L1-ORF1p-EGFP is coloured green. Visible are EGFP-enriched L1 bodies. Nuclei were stained with Hoechst and are marked by a cyan contour. White bar represents 9 μm. ( B ) Confocal microscopy z-stacks were analysed visually and the number of L1 bodies were quantified in 23 wild-type and 23 XRN1 KO cells. Median number of L1 bodies is shown as a line. ( C ) Result of an automated imaging flow cytometry analysis. All six wild-type and six XRN1 KO cell lines were transfected with the L1 -ORF1 -EGFP construct and the numbers of L1 bodies were calculated automatically. Normalized frequency adds up to 100%. Data are shown as mean ± SEM. The two fits were made and their statistically significant difference was estimated by F -test using Prism software. ( D ) Numbers of L1 bodies detected in the analysis in panel C. Median numbers are shown as black lines. Statistical significance between the two sets was calculated by a non-parametric Mann-Whitney test. ( E ) A graphical representation of the w orkflo w of the L1 amplification protocol (LEAP). Following transfection of the cells with L1 reporter the L1 bodies are pelleted by ultracentrifugation and combined with anchoring oligodT (dT 12 VN) primers to initiate re v erse transcription by L1-ORF2p. PCR with outer primers is used to amplify L1 fragments. ( F ) Results of the LEAP assay using wild-type (WT), XRN1 KO and DCP2 KO 293T cells as indicated. Control R T w as perf ormed by addition of a commercial reverse transcriptase. Control condition refers to blank ultracentrifugation (without cell lysate). Numbers on ladder bands indicate their DNA lengths. ( G ) Western blot to detect L1-ORF2p, L1-ORF1p, Lamin B and γH2AX in the cleared lysates and ultracentrifuged material (UC pellets) from the LEAP experiment presented in panel F. ( H ) Western blot analysis of cell lysates from six wild-type and six XRN1 KO cell lines f ollo wing transfection with the JM1 0 1 / L1.3 nomarker . Lamin B is loading control. ( I ) A quantitation of the ORF2p signal in panel G normalized to lamin B signal and with mean of the wild-types set to 1.00. Data are shown as mean ± SEM.

Article Snippet: Garcia-Perez), L1ORF2p (MT49 ( 68 ), kind gift of drs Kathleen Burns & Martin Taylor), γH2AX (Bethyl; A700-053-T), GFP (SantaCruz; sc-9996), PABPC1 (Proteintech; 10970-1-AP), TUT7 (Sigma; HPA020620), TUT4 (Proteintech; 18980-1-AP).

Techniques: Knock-Out, Confocal Microscopy, Transfection, Expressing, Construct, Staining, Imaging, Flow Cytometry, Software, MANN-WHITNEY, Amplification, Control, Reverse Transcription, Western Blot, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Stabilization of the methyl-CpG binding protein ZBTB38 by the deubiquitinase USP9X limits the occurrence and toxicity of oxidative stress in human cells

doi: 10.1093/nar/gky149

Figure Lengend Snippet: The ZBTB38 protein accumulates in H 2 O 2 -treated cells, and the accumulation depends on ROS and USP9X activity. ( A ) Western blot analysis in HeLa cells exposed to H 2 O 2 at the indicated concentrations for 30 min. P- H2AX: phosphorylated H2AX. Hydroxyurea treatment (HU) was used as a positive condition for H2AX phosphorylation. ( B ) ZBTB38 mRNA levels in samples from panel A. qRT-PCR normalized with three housekeeping genes (MAPK14, TBP and TFRC), n = 3. ( C ) Left panel: treatment of HeLa cells with Etoposide (Etop.; 200 μM for 1 h) or Hydroxyurea (HU; 2 mM for 2 h) causes DNA damage, but no ZBTB38 accumulation. Right panel: Etoposide or Hydroxyurea do not increase the intracellular ROS levels, as measured by DHE staining. The mean DHE intensity is indicated on the graph. ( D ) Extracellular Superoxide Dismutase (SOD) or Catalase (Cat.) do not prevent ZBTB38 accumulation in response to H 2 O 2 treatment (200 μM for 30 min), but PEG-coupled SOD or Cat., which are cell-permeable, do prevent ZBTB38 accumulation. ( E ) USP9X, but not USP7, accumulates in cells treated with H 2 O 2 (200 μM for 30 min). ( F ) ZBTB38 protein does not accumulate in response to H 2 O 2 in cells depleted of UPS9x. ( G ) Inhibition of USP9X by WP1130 prevents ZBTB38 accumulation in response to H 2 O 2 treatment.

Article Snippet: References of primary antibodies used in this study are: ZBTB38 (homemade antibody #148), USP9X (Millipore #MABE352; Abcam #19879; Bethyl Lab #A301-351A), GST (CST #2622), GAPDH (Abcam #ab9485), USP7 (Abcam #4080), HA (CST,#2367), H2AX (Bethyl Lab #A300-083A), phospho-H2AX (Millipore #05–636), TUBULIN (Abcam #ab7291), RFP (Rockland #600-401-379), V5 (Santa Cruz #sc-84594), MYC (CST #2276), WEE1 (Abcam #79298), NRF2 (Abcam #ab62352), PGC1 (Abcam #72230), DNMT1 (Imgenex #IMG-261A), ORC2 (Santa Cruz #28742), phospho-CHK1 (CST #2341), phospho-CHK2 (CST #2661), phospho-ATM (CST #4526P), PARP1 (CST #9542), JNK (Santa Cruz #474) and 53BP1 (Abcam #21083).

Techniques: Activity Assay, Western Blot, Phospho-proteomics, Quantitative RT-PCR, Staining, Inhibition

Journal: bioRxiv

Article Title: SMARCA2 and SMARCA4 participate in DNA damage repair

doi: 10.1101/2024.03.28.587286

Figure Lengend Snippet: The relocation of the SWI/SNF to DNA lesions. (A) The relocation of SMARCC1 and SMARCD1 at laser strip. (B) HeLa cells were pre-treated with or without 1 μM FHD286 for two hours. Laser microirradiation was performed to examine the localization of SMARCA2, SMARCC1 and SMARCD1. (C) HeLa cells were treated with 1 μM ATMi (ku55933), ATRi (VE-821), CBP/p300i (CBP/p300-IN-21) or PARPi (Olaparib) for two hours followed by laser microirradiation, the localization of SMARCA2, SMARCC1 and SMARCD1 were examined. The localization of the subunits of the SWI/SNF complex was also examined in the H2AX KO HeLa cells.

Article Snippet: The following antibodies were purchased from respective companies: anti-γH2AX (Novus, NB100-384), anti-RAD51 (Invitrogen, MA1-23271), anti-RNF8 (14112-1-AP, Proteintech), anti-BRCA1 (NB100-404, Novus), anti-SMARCA2 (Sigma, HPA029981).

Techniques: Stripping Membranes

Journal: bioRxiv

Article Title: SMARCA2 and SMARCA4 participate in DNA damage repair

doi: 10.1101/2024.03.28.587286

Figure Lengend Snippet: Lacking the enzymatic activities of SMARCA2/4 extends the accumulation of γH2AX, RNF8 and BRCA1. HeLa cells were pre-treated with or without FHD286 (1 μM) or Compound 14 (1 μM) for two hours followed by 5 Gy of IR. Following the indicated recovery time, the foci of γH2AX, RNF8 and BRCA1 were examined by IF. Statistical analyses were shown in the low panels.

Article Snippet: The following antibodies were purchased from respective companies: anti-γH2AX (Novus, NB100-384), anti-RAD51 (Invitrogen, MA1-23271), anti-RNF8 (14112-1-AP, Proteintech), anti-BRCA1 (NB100-404, Novus), anti-SMARCA2 (Sigma, HPA029981).

Techniques: