Journal: bioRxiv

Article Title: Imaging-Guided Metabolic Radiosensitization of Pediatric Rhabdoid Tumors

doi: 10.1101/2024.08.09.607364

Figure Lengend Snippet: (A) Whole tissue imaging and ( B ) confocal micrographs of γ-H2AX immunofluorescence staining of all treatment groups on Day 9. (Insets) Stained A-204 tissue at high magnification. Scale bar = 50 µm for confocal images and scale bar = 1000 µm for whole tumor sections. (C) Quantification of γ-H2AX staining in whole tumor sections ( a ) and confocal micrographs ( b ) of all treatment groups. (D) H&E staining for heart, liver, kidney and muscle from various treatment groups. Scale bar=100 µm. Data represented as mean ± SD.

Article Snippet: Alexa Fluor 488 AffiniPure Donkey Anti-Rat lgG(H+L) (Lot 163136) and Rhodamine (TRITC) AffiniPure Donkey Anti-Rat lgG(H+L) (Lot 157518) were obtained from Jackson ImmunoResearch Inc. Histone H2AX antibody (NB100-384) was purchased from Novus Biologicals and secondary antibody Donkey anti-Rabbit lgG (H+L) Dylight594 (SA5-10040) from Invitrogen.

Techniques: Imaging, Immunofluorescence, Staining

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Combining [ 177 Lu]Lu-DOTA-TOC PRRT with PARP inhibitors to enhance treatment efficacy in small cell lung cancer

doi: 10.1007/s00259-024-06844-1

Figure Lengend Snippet: Determination of γH2AX foci formation and clonogenic survival in H446 cells. (A) Exemplary images of H446 cells after mono- or combination therapy directly after treatment or after a 23 h post-incubation time. (B) Number of γH2AX foci per cell after mono- or combination therapy directly after treatment or after a 23 h post-incubation time in medium. Displayed are mean ± SEM from 3 individual experiments. (C) Surviving fractions of H446 cells mono- or combination therapy. Displayed are mean values from 2 independent experiments

Article Snippet: 1:150 dilution), γH2AX (pSER139, Novus #NB100-2280, 1:500 dilution) with optimized protocols of the Comparative Experimental Pathology Core Facility.

Techniques: Incubation

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Combining [ 177 Lu]Lu-DOTA-TOC PRRT with PARP inhibitors to enhance treatment efficacy in small cell lung cancer

doi: 10.1007/s00259-024-06844-1

Figure Lengend Snippet: Immunohistochemistry of H69 tumors. Exemplary stainings of H69 tumors obtained from the single dose PRRT study ( n = 1 animal/group) for SSTR2, PARP1, γH2AX (DNA damage marker) and cleaved caspase 3 (apoptosis marker) are displayed. Scale bar = 100 µm. Brown color indicates positivity for the respective marker

Article Snippet: 1:150 dilution), γH2AX (pSER139, Novus #NB100-2280, 1:500 dilution) with optimized protocols of the Comparative Experimental Pathology Core Facility.

Techniques: Immunohistochemistry, Marker

Journal: Nature Communications

Article Title: Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors

doi: 10.1038/s41467-024-46593-1

Figure Lengend Snippet: a Structures of Polθi. b Strand displacement assay (left). Scatter plot showing inhibition curve of MC160385. Data represent mean. n = 3 (technical replicates) +/-s.d. c Schematic of Polθ MMEJ. d Schematic of MMEJ DNA (top). Non-denaturing gel showing inhibition of Polθ-pol MMEJ by MC160385 (Bottom). % MMEJ indicated. n = 2 (performed twice). e Scatter plot showing relative velocity of dAMP incorporation by Polθ-pol in the presence of the indicated concentrations of MC160385. Data represent mean. n = 2 (technical replicates) +/- s.d. f Structures of improved Polθi. g Scatter plot showing inhibition curve of RTx-161. Data represent mean. n = 3 (technical replicates) +/- .s.d. h – l Scatter plots showing clonogenic survival following treatment with RTx-161 relative to DMSO. Data represent mean of 3 independent experiments ( n = 3 biological replicates) performed in triplicate ± SEM ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Two-sample t -test and P -values are indicated. h n = 3, P = 0.000102 for 5 μM, DLD1 BRCA2 -/- vs DLD1 Parental; i n = 3, P = 0.000091 for 4 μM HCT116 BRCA2 -/- vs HCT116 Parental; j n = 3, P = 0.001193 for 5 μM EUFA1341 PALB2 -/- vs EUFA1341 Parental; k n = 3, P = 0.004734 for 10 μM Brca1 CC/CC vs Brca1 -WT MEFs; l n = 2, P = 0.016209 for 10 μM Brca1 CC/CC vs Brca1 -WT MEFs, P = not significant for 10 μM Polq-/- vs Brca1 -WT MEFs. m Schematic of MMEJ DNA (top). Microhomology, red. Denaturing gel showing RTx-161 inhibition of Polθ-pol MMEJ (bottom). n = 1 (performed once). n Schematic of MMEJ reporter (left). Bar plot showing reduction in cellular MMEJ after treatment with 20 μM RTx-161 (right). Data represent mean from 3 biological replicates performed in triplicate. +/-s.d. n = 3, P < 0.0001 for 20 μM RTx-161. o Representative images of γ-H2AX phosphorylation following DMSO and RTx-161 treatment in DLD1 BRCA2 -/- or DLD1 Parental cells (left). Scatter plot shows percentage of nuclei with γ-H2AX foci. n = 3 (biological replicates), P = 0.000369 for RTx-161 treated, DLD1 BRCA2 -/- vs DLD1 Parental. p Western blot showing γ-H2AX phosphorylation and PARP cleavage following DMSO or RTx-161 treatment. GAPDH is shown as loading control. Source Data are provided as a Source data file.

Article Snippet: The primary antibody used for IF was rabbit anti-gamma H2AX [p Ser139] antibody (Bethyl Lab #A700-053) 1:500 dilution in 1% BSA in PBS.

Techniques: Inhibition, Phospho-proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Targeting NAD + Metabolism as Interventions for Mitochondrial Disease

doi: 10.1038/s41598-019-39419-4

Figure Lengend Snippet: Supplementation of dimethyl α-ketoglutarate (DMKG) extended lifespan and delays the onset of clasping in Ndufs4-KO mice. ( A ) Survival curves of WT mice, KO mice treated with vehicle (VEH), NMN or DMKG. N = 10–16. ( B ) Table summarizing median lifespan and clasping occurrence of Ndufs4-KO mice with vehicle, NMN or DMKG treatments. N = 10–16. Log-rank test was used. ( C ) Levels of SOD2 protein, SOD2 acetylation, H2Ax phosphorylation (H2Ax-Pi), and protein PAR in brain tissues in DMKG treatment cohort at P-50 were quantified. N = 3–6. Protein levels of ( D ) HIF1a and ( E ) LDHA in brain tissues were measured by Western blots. N = 3. Full blot images are presented in Supplementary Fig. . * P < 0.05 versus WT-VEH; # P < 0.05 versus KO-VEH. NS: not statistically significant versus KO-VEH. One-way ANOVA with Newman-Keuls multiple comparison test was used. SDHA and actin were used as loading control.

Article Snippet: Antibodies from the following companies were used for Western blot analysis: Phospho-H2Ax (1:500, NBP1-19255-Novus Biological), PAR (1:500, AM80100UG-Millipore), actin (1:5000, sc-8432-Santa Cruz), nitrotyrosine (1:500, 06-284-Millipore) acetyl-lysine (1:1000, 9441-Cell signaling), HIF1a (1:400, 14179-Cell Signaling), GLUT1 (1:500, ab652-Abcam), PDK4 (1:1000, 3820-Cell Signaling), LDHA (1:1000, 3582-Cell Signaling), SDHA (1:10000, ab14715-Abcam), Ndufs4 (1:1000, ab87399-Abcam,), SOD2 (1:1000, ab16956-Abcam) SOD2-Ac (1:1000, ab137037-Abcam), GDH (1:1000, 12793-Cell Signaling).

Techniques: Phospho-proteomics, Western Blot, Comparison, Control