Journal: bioRxiv

Article Title: Stepwise developmental mimicry generates proximal-biased kidney organoids

doi: 10.1101/2024.06.28.601028

Figure Lengend Snippet: a . Live imaging of HNF4A-YFP (Vanslambrouck et al., 2019 JASN ) kidney organoids on differentiation day 18 with Alexa 647-conjugated dextran uptake. Quantification of individual uptake events displayed in lower left, and insets detail HNF4A-YFP and dextran distinction. Scale bars: whole organoid-500 microns, insets-100 microns. b . Whole-mount immunofluorescence of day 18 control and proximal-biased kidney organoids following dextran uptake assay. Boxed region is magnified and split by individual channels. Scale bars: 10 microns. c . Whole-mount immunofluorescence of uninjured and 5 µM cisplatin-injured kidney organoids on differentiation day 21, split by control and proximal-biased conditions. Boxed area magnifies HNF4A + proximal organoid nephron regions. Scale bars: 10 microns. d . Timeline schematic of kidney organoid injury strategy. e . Quantification of relative protein signal from HAVCR1 + regions in control and proximal-biased kidney organoid nephrons on day 21 following injury. Data are averaged from n = 3 independent and consistent imaging panels. SEM error bars are shown. Statistical significance is determined using Student’s t test. f . Quantification of HNF4A + proximal organoid nephron segments with HAVCR1 expression for each of the four conditions from (c). Positive segments are counted from a 20X widefield panel of 4 images per organoid, from n = 2 biological replicate organoids for each condition. SEM error bars are shown. Statistical significance is determined using Student’s t test. g . Whole-mount immunofluorescence of uninjured and 5 µM cisplatin-injured kidney organoid nephrons on day 21. Dashed white line indicates an uninterrupted segment through the lumen of the organoid tubule. Scale bars: 10 microns. h . Quantification of continuous lumen segments from (h). Data are quantified from n = 4 organoids per condition, and SEM error bars are shown. Statistical significance is determined using Student’s t test. i . Whole-mount immunofluorescence of uninjured and 5 µM cisplatin-injured proximal-biased kidney organoid nephrons on day 21. Yellow arrows denote cells of the HNF4A + organoid nephron tubule with low HNF4A protein expression and positive expression of γH2AX. White arrows indicate cells with high HNF4A, and low γH2AX detection. Split channels separate HNF4A and γH2AX for cisplatin-injured condition. Scale bars: 10 microns. j . Whole-mount immunofluorescence of uninjured and 5 µM cisplatin-injured proximal-biased kidney organoid nephrons on day 21. White arrowheads indicate interstitial cells surrounding the tubules that are positive for SOX9. Scale bars: 10 microns. k-l . Quantification of HNF4A and SOX9 signal intensities from (j) for uninjured (k) and +cisplatin (l) samples. Intensities were quantified from individual cells of technical and biological duplicates (4 organoids per condition) and normalized using min-max normalization.

Article Snippet: Primary antibodies used in this study were: WT1 (abcam, ab89901, 1:1000), JAG1 (R&D Systems, AF599, 1:300), HNF1B (Thermo Fisher Scientific, MA5-24605, 1:500), HNF4A (R&D Systems, MAB4605, 1:200), CDH1 (BD Biosciences, 610181, 1:300), ZO-1 (Thermo Fisher Scientific, 33-9100, 1:200), PAX2 (R&D Systems, AF3364, 1:50), SIX1 (Cell Signaling Technology, 12891S, 1:300), HES1 (Cell Signaling Technology, 11988, 1:300), POU3F3 (Novus Biologicals, NBP1-49872, 1:500), HNF4G (Thermo Fisher Scientific, PA5-82189, 1:200), LRP2 (My Bio Source, MBS690201, 1:500), HAVCR1 (R&D Systems, AF1750, 1:200), γH2AX (Cell Signaling Technology, 2577), LAMB1 (Santa Cruz Biotechnology, sc-33709, 1:250), ATP1A1 (Abcam, ab7671, 1:200), and SOX9 (Abcam, ab185230, 1:300).

Techniques: Imaging, Immunofluorescence, Control, Expressing

Journal: Scientific Reports

Article Title: HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

doi: 10.1038/srep32960

Figure Lengend Snippet: ( a , b ) Binding of C6 -cell nuclear proteins to HMGB-protein affinity columns. Proteins in elution fractions were analyzed with silver stained SDS-PAGE. The bands, representing core histones, are indicted with marks on the left. a = HMGB1 –affinity column, b = HMGB4 –affinity column. ( c ) Core histones eluted from HMGB-protein affinity columns and histones were quantified with ELISA. Affinity chromatography was done as described above (n ≥ 3, *p < 0.05, ±SD; Ac = acetylated, 2Me = di-methylated, 3Me = tri-methylated (lysine)). ( d ) HMGB1 and HMGB4 differ in their histone H1 binding capacity. Affinity chromatography was done as described above and eluted proteins were analyzed with histone H1 ELISA. Elution peak areas were quantified (n = 3, ± SD, *p < 0.002). ( e ) HDACs bind to HMGB1 and HMGB4. Nuclear proteins were analyzed with affinity chromatography as described above except that eluted fractions from each column were pooled to form a single fraction. Relative HDAC and sirtuin activities in elution fractions were determined (n ≥ 3 in each experiment, ±SD). ( f ) Immunofluorescence intensities of modified histone antibody stained HMGB4 or HMGB4L1 overexpressing cells. Rat glioblastoma C6- cells transiently expressing HMGB4-V5 or HMGB4L1-V5 were double-immunostained with anti-acetylated (K9/K14), anti-dimethylated K27 or anti-trimethylated K27 histone H3 antibodies and with anti-V5 antibodies. The correlation blots are shown. ( g ) Regulation of protein levels in neuronal precursor cells by HMGB4 shRNA. Human neuronal precursor NTERA-2 cl. D1 cells stably overexpressing HMGB4 shRNA or control nonspecific shRNA were analyzed in cell ELISA. Absorbance values of HMGB4 shRNA expressing cells were normalized to values of control shRNA expressing cells (n ≥ 3, ± SD, *p < 0.03). H2A K5 Ac = histone H2A acetylated lysine 5, H4 K8 Ac = histone H4 acetylated lysine 8.

Article Snippet: The following antibodies were used in cell ELISA: mouse anti-β-Actin (clone AC-15; Sigma-Aldrich), mouse anti-GFAP (clone G-A-5; Sigma-Aldrich), mouse anti-NCAM (clone 5B8; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), rabbit anti-ASCL1 (Biobryt Ltd., Cambridgeshire, UK), rabbit anti-human HMGB4 (Boster, Pleasanton, CA, USA), rabbit anti-histone H2A acetylated Lys5 and rabbit anti-histone H4 acetylated Lys8 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Binding Assay, Staining, SDS Page, Affinity Column, Enzyme-linked Immunosorbent Assay, Affinity Chromatography, Methylation, Immunofluorescence, Modification, Expressing, shRNA, Stable Transfection, Control, In-Cell ELISA

Journal: Scientific Reports

Article Title: HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

doi: 10.1038/srep32960

Figure Lengend Snippet: Biological processes associated with HMGB4 expression.

Article Snippet: The following antibodies were used in cell ELISA: mouse anti-β-Actin (clone AC-15; Sigma-Aldrich), mouse anti-GFAP (clone G-A-5; Sigma-Aldrich), mouse anti-NCAM (clone 5B8; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), rabbit anti-ASCL1 (Biobryt Ltd., Cambridgeshire, UK), rabbit anti-human HMGB4 (Boster, Pleasanton, CA, USA), rabbit anti-histone H2A acetylated Lys5 and rabbit anti-histone H4 acetylated Lys8 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Over Expression

Journal: Nature

Article Title: TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein

doi: 10.1038/nature13955

Figure Lengend Snippet: List of primers used for quantitative real-time RT-PCR, ChIP and vector construction.

Article Snippet: Proteins were resolved by 15% SDS-PAGE and subjected to immunoblotting with H2A-ub(K119) (Cell Signaling Technology #8240, clone D27C4), H2A (Cell Signaling Technology #2578), H2B-ub (EMD Millipore 05-1312, clone 56), H2B (Abcam ab18977), H3 (Cell Signaling Technology #9715) or H4 (Cell Signaling Technology #2592) antibodies.

Techniques: Quantitative RT-PCR, Plasmid Preparation, Cloning

Journal: Molecules (Basel, Switzerland)

Article Title: Antitumoral Activity of the Universal Methyl Donor S -Adenosylmethionine in Glioblastoma Cells.

doi: 10.3390/molecules29081708

Figure Lengend Snippet: Figure 5. Effects of AdoMet on DNA damage response in GBM cells. (A) U251MG, U87MG, and U343MG cells were cultured for 72 h in medium supplemented or not (control) with AdoMet 500 µM. The levels of the main proteins involved in DNA repair and DNA damage response were then evaluated by Western blot, and the relative densitometric analyses, performed for each protein in relation to its relative housekeeping included in the Supplementary Figures S7–S9 (showing the uncropped images of Western blots), are reported as a percentage of untreated cells (100%). Representative housekeeping β-actin protein, used as a loading control, was reported. Error bars represent the SD. * p < 0.05, ** p < 0.01, *** p < 0.005 versus untreated cells. (B) Effect of AdoMet on γH2AX and RAD51 foci formation. Representative images of immunofluorescence staining for phosphorylated γH2AX (red) and RAD51 (green) in U343MG cells treated or not (control) with AdoMet. The overlapping of the two signals (merge panel) in AdoMet-treated cells evidenced the reduced expression of RAD51 and the concomitant increase in γH2AX foci. Hoechst 33258 was used for nuclear staining (blue). Scale bar: 20 µm. About 100 nuclei for each group were scored in each experiment, and a threshold of five foci per cell was considered positive and reported in the histograms as a mean of γH2AX and RAD51 foci. Values represent the means of three experiments ± SD (* p < 0.05, ** p < 0.01).

Article Snippet: Monoclonal antibodies (mAb) to Chk1 (sc-8408), BRCA1 (sc-1021), histone H2AX (sc-517336), p-Histone H2AX (sc-517348) and β-actin (sc-47778) were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Control, Western Blot, Immunofluorescence, Staining, Expressing

Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 −/− ESCs are impaired in ATM activation and hypersensitive to DOX treatment. (A) Phosphorylation of ATM, H2AX, and p53 in Sall4 +/− and Sall4 −/− ESCs at different time points after DOX treatment. (B and C) Sall4 +/− and Sall4 −/− ESCs were mixed, treated with DOX or mock treated, and examined for the foci formation by ATM-Ser1987p (B) or γ-H2AX (C). DNA is counterstained with DAPI (blue). Bars, 10 µm. Graphs show the change of average fluorescence intensity (AFI) determined by dividing the overall mean fluorescence intensity by the area of the cell, and values are mean ± SEM. **, P < 0.01; ***, P < 0.001 by t test throughout the figure. (D) Cell cycle G 2 /M checkpoint is impaired in Sall4 −/− ESCs after DOX treatment. (top) Mock-treated control. (bottom) 1 h after after DOX treatment. The values are means ± SEM ( n = 4). *, P < 0.05 by t test. Circles denote the percentages of cells positive for histone 3 phosphorylated at Ser10 in all cells. PI-A, propidium iodide area. (E) Inducible expression of Sall4 in Sall4 −/− ESCs rescues the activation of ATM after DOX (0.5 µM) treatment. Sall4 −/− ESCs were stably transfected with a doxycycline-inducible vector expressing ectopic Sall4 (Lenti- Sall4 ).

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Activation Assay, Phospho-proteomics, Fluorescence, Control, Expressing, Stable Transfection, Transfection, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 is relocated from the heterochromatin to DNA DSBs in ESCs after DOX treatment. (A) Sall4 is sequestered in the heterochromatin and relocated to DNA DSBs in ESCs after DNA damage. Sall4 +/− cells were examined for the foci formation of γ-H2AX and Sall4 before and after DNA DSB damage induced by DOX. Nuclei were counterstained with DAPI (blue). For intensity correlation analysis (ICA), pixels from the Sall4 channel covarying positively with the corresponding signal from the γ-H2AX or DAPI channel are denoted. Bar, 5 µm. (B) Rr analysis for the colocalization of Sall4 and γ-H2AX. Rr is the Pearson’s correlation coefficient, and a value of 1 indicates perfect colocalization. The values of Rr are means ± SEM. ***, P < 0.001 by t test. (C) Sall4 is colocalized with HP1-α foci before DNA DSB damage and is mobilized out of HP1-α foci after DNA damage. Nuclei are counterstained with DAPI (blue). Bar, 5 µm. (D) The average fluorescence intensity (AFI) of Sall4 at heterochromatin was determined by dividing the overall mean fluorescence intensity by the area of the cell, and values are means ± SEM. **, P < 0.01 by t test. (E) DNA damage disrupts the interaction between Sall4 and HP1-α in ESCs. The interaction between Sall4 and HP1-α was analyzed by coimmunoprecipitation (Co-IP) with the HP1-α antibody.

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Fluorescence, Co-Immunoprecipitation Assay

Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 linking SWI/SNF-associated chromatin remodeling to MRN-dependent ATM activation. (A) Silencing of Baf60a impairs the recruitment of Sall4 to γ-H2AX foci after DNA DSB damage. Nuclei are counterstained with DAPI (blue). For intensity correlation analysis (ICA), pixels from the Sall4 channel covarying positively with the corresponding signal from the γ-H2AX channel are denoted. si-Con, control siRNA. Bar, 5 µm. (B) Inhibition of H3K14ac by CPTH2 disrupts the association of Baf60a, Sall4, and ATM with DNA DSBs in the cellular extract of Sall4 +/− ESCs treated with 0.5 µM DOX for 4 h. WNE, whole nuclear extract. (C) Inhibition of H3K14ac by CPTH2 in Sall4 +/− ESCs impaired ATM-dependent responses to DNA DSB damage. (D) The kinetics of the recruitment of Rad50, Sall4, and Balf60a to the specific DNA DSB. ChIP analysis showed the kinetics of the recruitment of Sall4, Rad50, and Baf60a to the single I-PpoI cleavage site at chromosome 3 in Sall4 +/− cells. Time denotes the minutes after the addition of 4-hydroxytamoxifen. Values are means ± SEM. (E) An illustrated model for Sall4 to function as a transducer of chromatin remodeling to ATM signaling in ESCs after DNA damage. P, phosphorylation.

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Activation Assay, Control, Inhibition, Phospho-proteomics