Journal: Frontiers in Oncology

Article Title: Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

doi: 10.3389/fonc.2021.612354

Figure Lengend Snippet: HSP90i by NW457 leads to downregulation of DNA damage response factors, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in human glioblastoma cells. (A) Transcriptomic profiling of regulators of the DNA damage response (DDR) in LN229 and T98G cells. mRNA expression levels were determined by qRT-PCR, normalized to a matrix of 3 reference genes (18S rRNA, δ-amino-laevulinate-synthase, and β2-microglobulin), and calibrated to the results of untransformed human astrocytes. For both cell lines, three replicates were analyzed and are displayed as x-fold log2-values. (B) Time course analysis of DDR regulator protein expression in LN229 and T98G cells upon HSP90i by 10 nM NW457. Arrowheads indicate the bands that were used for quantification. Protein levels were normalized to a matrix comprising vinculin and α-tubulin and are depicted as x-fold log2-values compared to the 0 h controls. (C) Immunofluorescence microscopy of γH2AX and 53BP1 DNA damage repair foci in LN229 and T98G cells upon irradiation at 2 Gy ± HSP90i by NW457. Cells were treated with NW457 (10 nM) or DMSO for 24 h, irradiated, and fixed at the indicated times. Cells were stained for γH2AX, 53BP1, and DNA and subjected to deconvolution immunofluorescence microscopy. Scale bar depicts 10 µm. (D) Quantification of DNA damage repair kinetics from (C) . γH2AX and 53BP1 double-positive foci in at least 20 randomly picked nuclei were counted by hand. Individual data points with superimposed means ± 95% confidence intervals are displayed, and overall curve comparison was performed by two-way ANOVA. (E) Clonogenic survival of LN229 and T98G cells upon irradiation at 0–8 Gy ± HSP90i by NW457. Cells were pre-treated with 10 nM NW457 or DMSO for 24 h followed by irradiation at the indicated doses, and colony formation was allowed for 13 d ± continuous NW457 treatment. Individual data points of 4 independent experiments are shown, linear-quadratic regression lines are superimposed, and overall curve comparison was performed by two-way ANOVA.

Article Snippet: Cells were stained with monoclonal mouse anti-γH2AX (Merck Millipore) and polyclonal rabbit anti-53BP1 (Bio-Techne, Wiesbaden, Germany) antibodies diluted in 3% isotonic bovine serum albumin and 0.1% Triton X-100 for 2 h at room temperature.

Techniques: Irradiation, Expressing, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining, Comparison

Journal: Genes & Development

Article Title: Replication stress conferred by POT1 dysfunction promotes telomere relocalization to the nuclear pore

doi: 10.1101/gad.337287.120

Figure Lengend Snippet: The NUP62 subcomplex is necessary to maintain telomere integrity in cells carrying mutant POT1. (A) Schematic of the nuclear pore complex in mammalian cells (Kabachinski and Schwartz 2015). Highlighted in bold are SL hits with POT1 mutants and in red are nucleoporins (NUPs) enriched in the POT1-ΔOB BioID IPs. NUP153 and TPR were hits in both screens. (B) Incucyte growth analysis of RPE-1 p53−/− cells expressing POT1-WT and POT1-ΔOB, treated with shRNAs against NUP62, NUP58, and scramble control. Cell proliferation was monitored over 160h. Graph representing data from two independent experiments. (C) Representative images displaying telomere dysfunction-induced foci (TIFs) in RPE-1 p53−/− cells expressing POT1-ΔOB and treated with shRNAs against NUP58 and NUP62, as well as control shRNA. 53BP1 in red is detected by indirect immunofluorescence, and telomeres are marked with FISH in green. DNA is counterstained with DAPI in blue. (D) Quantification of the percentage of cells with five or more TIFs in RPE-1 p53−/− cells with the indicated treatment. Graph represents the mean of n = 3 independent experiments with SD (two-tailed t-test). (E,F) Analysis of telomere fragility in RPE-1 p53−/− cells expressing POT1-WT and POT1-ΔOB and treated with the indicated shRNA. (E) Representative images showing metaphase chromosomes from POT1-ΔOB cells with fragile telomeres. Arrowheads indicate examples of fragile telomeres on the metaphase. Telomeres were stained with FISH in green, while DNA is detected with DAPI in blue. (F) Quantification of fragile telomeres per metaphase in cells with the indicated treatments. Graph represents mean of four independent experiments (n > 40 metaphase per condition) with SD (one-way ANOVA test). (G,H) Telomere sister chromatid exchange (T-SCE) analysis in cells with the indicated treatment. (G) Image depicting metaphase chromosomes with T-SCE events (white arrows). Telomeres were stained with FISH in green and red and DNA is counterstained with DAPI in blue. (H) Quantification of T-SCE events per metaphase in the indicated cells (n > 15 metaphases per condition).

Article Snippet: The primary antibodies used were 53BP1 (1:1000; rabbit polyclonal; Novus Biologicals 100-304A) and Myc (1:1000; Cell Signaling 9B11).

Techniques: Mutagenesis, Expressing, shRNA, Immunofluorescence, Two Tailed Test, Staining