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: 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