Journal: Journal of Medicinal Chemistry

Article Title: RTx-303, an Orally Bioavailable Polθ Polymerase Inhibitor That Potentiates PARP Inhibitors in BRCA Mutant Tumors

doi: 10.1021/acs.jmedchem.5c00551

Figure Lengend Snippet: Cellular and in vitro activity of RTx-284. A. Bar plot showing the IC50 of RTx-284 in the indicated HR-proficient (gray) and HRD (red) cell lines. Data represent mean of at least 3 independent experiments performed in triplicate. B-D. Synergy plots created by Combenefit showing synergistic activity between RTx-284 and indicated PARPi in the indicated HRD cell lines. E. Bar plot showing that 10 μM RTx-284 suppresses the MMEJ GFP reporter. Data represent mean of 3 individual experiments performed in triplicate ±SEM. F. Dot plot showing increase of γH2AX following RTx-284:olaparib treatment in DLD1 BRCA2–/– cells. G. Bar plot showing that RTx-284 promotes an increase in caspase positive DLD1 BRCA2–/– cells. Data represent mean of 3 independent experiments performed in triplicate, ±SEM.

Article Snippet: Cells were incubated with primary antibody (rabbit antigamma H2AX (p Ser139) antibody, Bethyl Lab #A700–053, 1:500 dilution in 1% BSA in PBS) overnight at 4 °C followed by 3x washes with PBS and then 1 h incubation with secondary antibody (Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 488 (Thermo #35552) 1:2000 dilution in 1% BSA in PBS).

Techniques: In Vitro, Activity Assay

Journal: Journal of Thoracic Disease

Article Title: Role of PLEKHA7 in promoting radioresistance in esophageal cancer cells via the inhibition of cuproptosis

doi: 10.21037/jtd-2025-858

Figure Lengend Snippet: IR induces cuproptosis, and cuproptosis inducers increase radiosensitivity. (A,B) Bar graph showing the relative levels of total ROS by DCFDA staining in the indicated cells. (C,D) Diagrams showing the copper levels at 4 h post-IR. (E,F) Western blot showing the expression levels of proteins related to cuproptosis at different times after IR. (G,H) Colony formation assays of ECA-109 (G) and KYSE-150 (H) cells subjected to the indicated treatments. The cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted with ImageJ. Magnification times: 8×. (I,J) EdU assays of ECA-109 (I) and KYSE-150 (J) cells subjected to the indicated treatments. In accordance with the manufacturer’s instructions, cell viability was determined using a Cell-Light EdU Apollo567 In Vitro Kit. The results were captured using a fluorescence microscope. Scale bar: 100 μm. (K,L) Representative immunofluorescence images showing phospho-H2AX foci (green) and nuclei counterstained with DAPI (blue) in the indicated cells. Scale bar: 20 μm. All the experiments were repeated at least three times. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. DAPI, 4’,6-diamidino-2’-phenylindole; DCFDA, 2,7-dichlorodi -hydrofluorescein diacetate; DMSO, dimethyl sulfoxide; EdU, 5-ethynyl-2’-deoxyuridine; ES, elesclomol; IR, ionizing radiation; qPCR, quantitative polymerase chain reaction; ROS, reactive oxygen species; TTM, tetrathiomolybdate.

Article Snippet: The cells were then incubated with a p-Histone H2AX (S139) rabbit monoclonal antibody (CST, USA, 2577S, 1:200) overnight at 4 °C.

Techniques: Staining, Western Blot, Expressing, In Vitro, Fluorescence, Microscopy, Immunofluorescence, Real-time Polymerase Chain Reaction

Journal: Journal of Thoracic Disease

Article Title: Role of PLEKHA7 in promoting radioresistance in esophageal cancer cells via the inhibition of cuproptosis

doi: 10.21037/jtd-2025-858

Figure Lengend Snippet: PLEKHA7 confers radioresistance in a cuproptosis-dependent manner. (A,B) Western blot analysis of cuproptosis-related protein expression in ESCC cells subjected to the indicated treatments. (C,D) Intracellular copper levels in cells subjected to the indicated treatments. (E,F) Clonogenic survival assays of cells subjected to the indicated treatments. The cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted with ImageJ. Magnification times: 8×. (G,H) EdU assays were performed on ECA-109 (G) and KYSE-150 (H) cells subjected to the indicated treatments. In accordance with the manufacturer’s instructions, cell viability was determined using a Cell-Light EdU Apollo567 In Vitro Kit. The results were captured using a fluorescence microscope. Scale bar: 100 μm. (I,J) Representative immunofluorescence images showing phospho-H2AX foci in the indicated cells. Scale bar: 20 μm. All the experiments were repeated at least three times. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. DAPI, 4’,6-diamidino-2’-phenylindole; DMSO, dimethyl sulfoxide; EdU, 5-ethynyl-2’-deoxyuridine; ESCC, esophageal squamous cell carcinoma; IR, ionizing radiation; TTM, tetrathiomolybdate.

Article Snippet: The cells were then incubated with a p-Histone H2AX (S139) rabbit monoclonal antibody (CST, USA, 2577S, 1:200) overnight at 4 °C.

Techniques: Western Blot, Expressing, Staining, In Vitro, Fluorescence, Microscopy, Immunofluorescence

Journal: Journal of Thoracic Disease

Article Title: Role of PLEKHA7 in promoting radioresistance in esophageal cancer cells via the inhibition of cuproptosis

doi: 10.21037/jtd-2025-858

Figure Lengend Snippet: Elesclomol-CuCl 2 significantly enhances the radiosensitivity of PLEKHA7 -deficient cells. (A,B) Clonogenic survival assays of cells with the indicated transfections and treatments. The cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted with ImageJ. Magnification times: 8×. (C,D) EdU assays of cells subjected to different transfections and combined treatments with IR and elesclomol-CuCl 2 . In accordance with the manufacturer’s instructions, cell viability was determined using a Cell-Light EdU Apollo567 In Vitro Kit. The results were captured using a fluorescence microscope. Scale bar: 100 μm. (E,F) Representative immunofluorescence images showing staining of phospho-H2AX foci in transfected cells treated with IR or elesclomol-CuCl 2 . Scale bar: 20 μm. All the experiments were repeated at least three times. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. DAPI, 4’,6-diamidino-2’-phenylindole; EdU, 5-ethynyl-2’-deoxyuridine; IR, ionizing radiation; ES, elesclomol.

Article Snippet: The cells were then incubated with a p-Histone H2AX (S139) rabbit monoclonal antibody (CST, USA, 2577S, 1:200) overnight at 4 °C.

Techniques: Transfection, Staining, In Vitro, Fluorescence, Microscopy, Immunofluorescence