Journal: Cancer Discovery

Article Title: Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers

doi: 10.1158/2159-8290.CD-24-0027

Figure Lengend Snippet: Translating RMC-6236 activity in NSCLC. A, Efficacy of RMC-6236 on Kras G12C , Kras G12D , Kras G12V , Kras G12A , Kras G13D , or Kras Q61H -driven autochthonous lung tumors in immunocompetent mice. A pool of lentiviral cDNA vectors encoding each oncogenic Kras variant was delivered intratracheally to the lungs of each mouse, and 13 weeks after tumor growth, mice were treated with RMC-6236 at 20 mg/kg po qd for 3 weeks prior to analysis. 95% confidence intervals are shown. B, Efficacy of RMC-6236 and adagrasib in the LUN055 NSCLC PDX model with KRAS G12C allele copy-number gain. Immunoblot Western analyses (left) of RAS and KRAS protein levels in NCI-H358 ( KRAS G12C/WT , NSCLC), LU99 ( KRAS G12C/WT , NSCLC), NCI-H2122 ( KRAS G12C/G12C , NSCLC), and LUN055 ( KRAS G12C/WT , NSCLC) xenograft tumors. Relative copy-number (middle) of KRAS WT or KRAS G12C in LUN055 xenograft tumors ( n = 2) were determined by ddPCR and normalized to ACTB . LUN055 xenograft tumor-bearing mice were treated with vehicle or RMC-6236 at 25 mg/kg po qd or adagrasib at 100 mg/kg po qd for 24 to 28 days ( n = 3 per group, right). Mean tumor volumes of each group were plotted over the course of treatment. Dotted line indicates the initial average tumor volume. Error bars, SEM. C, Efficacy of RMC-6236 in the intracranially implanted LU99-Luc ( KRAS G12C/WT , NSCLC) xenograft model ( n = 8 per group). RMC-6236 was dosed at 25 mg/kg daily for 21 days. Images of bioluminescence in individual mice were shown. Bioluminescence of ROI in vehicle control and RMC-6236 groups were compared by two-way repeated-measures ANOVA at day 21 (**, P < 0.01). Results were shown as mean ± SEM. D, Antitumor activity of RMC-6236 and the combination with anti–PD-1 (clone RMP1-14, rat IgG2a) following repeated administration in BALB/c mice bearing the murine colon carcinoma eCT26 ( Kras G12C/G12C ) shown as individual tumor growth curves ( n = 10 per group). Graphs indicate the number of complete regressions per injected mice. RMC-6236 and anti–PD-1 treatment started on day 17 after implantation. RMC-6236 treatment was stopped at day 31 after implantation and anti–PD-1 at day 35 after implantation. E, Antitumor activity of RMC-6236 following repeated administration in NSG mice bearing the murine colon carcinoma eCT26 ( Kras G12C/G12C ) shown as individual tumor growth curves ( n = 10 per group). Graphs indicate the number of complete regressions per injected mice. RMC-6236 treatment started on day 16 after implantation. F, Immune cell composition (CD8 + and CD4 + T cells, Ly6C + and Ly6G + myeloid-derived suppressor cells and M2 macrophages) in murine colon carcinoma eCT26 syngeneic tumors ( Kras G12C/G12C ) represented as percentage of CD45 + cells and expression of cell-surface markers on viable, CD45 − large cells (assessed as tumor cells) 24 hours post 4 days of treatment with vehicle or RMC-6236 at 25 mg/kg po qd n = 3 biological replicates/group represented as mean; *, P < 0.05; **, P < 0.01; ns, nonsignificant by two-sided Student t test.

Article Snippet: Probes and primers for the following genes were included in the multiplexed ddPCR: KRAS WT [dPCR Mutation Detection Assay KRAS Wild-Type for p.G12C, Human (apexbio, AA100902-WT)], KRAS G12C [dPCR Mutation Detection Assay KRAS Mutant for p.G12C, Human (apexbio, AA100902-MU)], and ACTB ( ACTB probe, 5′-Cy5-ATTGCCGACAGGATGCAGAAGGA-3′; ACTB primer F, 5′-GACATCCGCAAAGACCTGTA-3′; ACTB primer R, 5′-GGAAAGACACCCACCTTGAT-3′).

Techniques: Activity Assay, Variant Assay, Western Blot, Control, Injection, Derivative Assay, Expressing

Journal: British Journal of Cancer

Article Title: Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells

doi: 10.1038/sj.bjc.6604083

Figure Lengend Snippet: Influence of PHA680632 on cell cycle in p53wt vs p53−/− HCT116 cells. ( A and B ) analysis of the cell cycle. ( A ) Quantitative data of cell cycle distribution after PHA680632 and 6 Gy of irradiation in p53wt HCT116 (above) and p53−/− HCT116 (below) have been shown in the two histograms. The mean values (percentage of sub-population of different cell cycle: sub-G1, G1, S, G2–M, and >4 N cells is shown in different conditions: control, IR, PHA680632, or PHA680632+IR combination) of three independent experiments are shown and bar errors represent s.e.m. Twenty-four hours exposure to 400 n M PHA680632 led to the apparition of >4 N DNA content cells in the two HCT116 cell lines ( P =0.0081 and P =0.0005 for p53wt and p53−/− HCT116, respectively), PHA680632 induced a greater accumulation of cells with >4 N DNA content in p53−/− HCT116 cell line when compared to their p53 wild counterparts ( P =0.0482); a moderate G2–M block was observed 24 h after 6 Gy irradiation in the control cells. At 24 h after 6 Gy irradiation and PHA680632, exposure dramatically reduced the percentage of cells with >4 N DNA content cells compared with PHA680632 alone ( P =0.0068 and P =0.0119 for p53wt and p53−/− HCT116, respectively). ( B ) A representative cell cycle analysis in p53−/− HCT116 cells. ( C ) Immunofluorescence images showing phospho-T288-Aurora-A (Pho-Aurora-A) in mitotic p53wt HCT116 cells after 24 h exposure of 1 μ M PHA680632 (below) or control (above). β -Tubulin and Hoechst were used to visualise microtubule and DNA, respectively.

Article Snippet: HCT116 human colorectal cancer cell lines (wild-type (wt), p53−/−) ( Bunz et al , 1998 ) were a kind gift by B Vogelstein (Johns Hopkins, Baltimore) HT29 colorectal cancer cell line (p53 mutated) and A549 human non-small cell lung cancer (wild-type p53) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Irradiation, Control, Blocking Assay, Cell Cycle Assay, Immunofluorescence

Journal: British Journal of Cancer

Article Title: Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells

doi: 10.1038/sj.bjc.6604083

Figure Lengend Snippet: Influence of PHA680632 on clonogenic survival in different cancer cell lines. Clonogenic survival assay of cell lines exposed to a concentration range of PHA680632. ( A ) p53−/− HCT116; ( B ) p53wt HCT116; ( C ) HT29 (p53 mutated); ( D ) A549 (p53 wt).

Article Snippet: HCT116 human colorectal cancer cell lines (wild-type (wt), p53−/−) ( Bunz et al , 1998 ) were a kind gift by B Vogelstein (Johns Hopkins, Baltimore) HT29 colorectal cancer cell line (p53 mutated) and A549 human non-small cell lung cancer (wild-type p53) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Clonogenic Cell Survival Assay, Concentration Assay

Journal: British Journal of Cancer

Article Title: Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells

doi: 10.1038/sj.bjc.6604083

Figure Lengend Snippet: Influence of PHA680632 on the cellular response to irradiation in p53wt vs p53−/− HCT116 cells. ( A ) p53-dependent effect of the PHA680632 on clonogenic survival after irradiation; the cells were exposed to 100 n M PHA680632 for 24 h and then irradiated. Data represent the mean of three independent experiments in triplicate, and error bars represent s.d. for p53wt (left) and p53−/− (right) HCT116 cells. The surviving fraction after drug exposure+irradiation is normalised to survival for the same cells treated with the drug alone in the absence of irradiation (plating efficiency: 68.7 and 87%, respectively, for p53wt and p53−/− HCT116 cells exposed to 100 n M PHA680632 alone). For the two HCT116 cell lines, the P -values for the clonogenic survival were: P <0.0001 for DMSO vs PHA norm ctrl, P <0.0001 for DMSO vs PHA in p53wt HCT116, and P =0.0806 for DMSO vs PHA in p53−/− HCT116. ( B ) Cytofluorimetric detection of apoptotic parameters in p53 wt (left) and p53−/− (right) HCT116 cells, respectively, exposed to 100 n M for 24 h PHA680832 and then irradiated (6 Gy); 72 h after irradiation, cells were stained with Annexin V and PI and analysed by FACS. Quantification of the data were obtained; error bars represent s.d. For p53−/− HCT116, P -value was <0.0001 when comparing PHA680632 alone vs IR+PHA680632 and for IR alone vs IR+PHA680632 ( P <0.0001).

Article Snippet: HCT116 human colorectal cancer cell lines (wild-type (wt), p53−/−) ( Bunz et al , 1998 ) were a kind gift by B Vogelstein (Johns Hopkins, Baltimore) HT29 colorectal cancer cell line (p53 mutated) and A549 human non-small cell lung cancer (wild-type p53) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Irradiation, Staining

Journal: British Journal of Cancer

Article Title: Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells

doi: 10.1038/sj.bjc.6604083

Figure Lengend Snippet: Influence of Aurora-A inhibition by siRNA in response to IR in p53wt vs p53−/− HCT116 cells. ( A ) Western blot showing expression of Aurora-A 24 h after siRNA Aurora-A transfection compared with the non-specific targeting siRNA control; clonogenic survival of p53wt (left) or p53−/− (right) HCT116 cells transfected by siRNA Aurora-A or siRNA control; 24 h after transfection, cells were irradiated at indicated doses. Data represent the mean of three independent experiments in triplicate, and error bars represent s.d. ( B and C ) Aurora-A inhibition by siRNA influence on IR-induced micronuclei ( B ) and Brca1 foci formation ( C ) in p53wt HCT116 and p53−/− HCT116 cell lines. The percentage of cells with micronuclei (24 h after irradiation) in the p53−/− and p53wt HCT116 cells transfected by siRNA Aurora-A (siA) or siRNA control (siC) and then irradiated to 6 Gy is represented ( B ). There is a significant difference between siA 6 Gy and siC 6 Gy in p53−/− HCT116 ( P =0.0018), while no difference was found in p53wt HCT116 ( P =0.5102). No difference has been observed in siA and siC without irradiation ( P =0.8746 for p53wt HCT116 and P =0.614 for p53−/− HCT116). Immunofluorescent images of micronuclei after 6 Gy (or 0 Gy) in p53wt HCT116 cells ( B ). The mean BRCA1 foci (4 h after irradiation) number per cell are shown ( C ) in the p53−/− and p53wt HCT116 cells after transfection by siRNA Aurora-A (siA) or siRNA control (siC) and IR (24 h after transfection of siRNA, 6 Gy). Representative fluorescence microphotographs (Brca1 foci after 6 or 0 Gy) are shown in p53wt HCT116 cells.

Article Snippet: HCT116 human colorectal cancer cell lines (wild-type (wt), p53−/−) ( Bunz et al , 1998 ) were a kind gift by B Vogelstein (Johns Hopkins, Baltimore) HT29 colorectal cancer cell line (p53 mutated) and A549 human non-small cell lung cancer (wild-type p53) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Western Blot, Expressing, Transfection, Control, Irradiation, Fluorescence

Journal: British Journal of Cancer

Article Title: Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells

doi: 10.1038/sj.bjc.6604083

Figure Lengend Snippet: In vivo tumour growth delay after PHA680632 and irradiation. Left: in vivo experiments in p53−/− HCT116 subcutaneous xenograft. Animals were randomly assigned to control i.p. b.i.d. for 4 days with vehicle (control), PHA-680632 (PHA) 40 mg kg −1 alone b.i.d. for 4 days, IR alone 8 Gy in 1 day or a combination of both (IR began just after the second administration of PHA680632); n =6 per group, mean tumour volumes±s.e.m. are shown. The respective P -values for control vs IR+PHA, PHA vs IR+PHA and IR vs IR+PHA were <0.0001, 0.0003, and 0.0685 respectively. Right diagrammatic representation showing the mechanistic link between Aurora-A, p53, and irradiation.

Article Snippet: HCT116 human colorectal cancer cell lines (wild-type (wt), p53−/−) ( Bunz et al , 1998 ) were a kind gift by B Vogelstein (Johns Hopkins, Baltimore) HT29 colorectal cancer cell line (p53 mutated) and A549 human non-small cell lung cancer (wild-type p53) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo, Irradiation, Control

Journal: The Journal of Biological Chemistry

Article Title: Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing *

doi: 10.1074/jbc.M114.604959

Figure Lengend Snippet: Primers used in this study

Article Snippet: The wild type, enzymatically overactivated or inactivated mouse Ape1 was amplified by PCR using plasmid pCMV6-kan/neo mAPEX1 (purchased from Origene Inc.) as template with proper primers ( ).

Techniques: Sequencing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing *

doi: 10.1074/jbc.M114.604959

Figure Lengend Snippet: Up-regulation of Ape1 results in more DNA signals that were detected in the Mre11 immunoprecipitated complex in high LET-irradiated cells. A, the outline of a flow chart to detect DNA fragments in the Mre11-DNA complex. B, image of the DNA levels in the Mre11-DNA complex from non-irradiated (NR), low LET-irradiated, or high LET-irradiated Ogg1−/− MEF cells co-transfected with the control vector (Ogg1−/−) or the vector encoding Ogg1 (Ogg1+) with another control plasmid (CP) or the plasmid encoding Ape1. One-tenth of the immunoprecipitation (IP) samples were used as the internal loading control. C, quantitation of the image data representing the mean ± S.D. (error bars) from three independent experiments. **, p < 0.01.

Article Snippet: The wild type, enzymatically overactivated or inactivated mouse Ape1 was amplified by PCR using plasmid pCMV6-kan/neo mAPEX1 (purchased from Origene Inc.) as template with proper primers ( ).

Techniques: Immunoprecipitation, Irradiation, Transfection, Plasmid Preparation, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing *

doi: 10.1074/jbc.M114.604959

Figure Lengend Snippet: Up-regulation of Ape1 sensitizes cells to high LET radiation-induced killing. A, the Ape1 levels were detected by immunoblotting at 30 h after transfecting the Ape1-containing vector or control vector into the MEF cell lines. WT, wild type MEF cells; NHEJd, NHEJ-deficient Ku80−/− MEF cells. β-Actin was used as the internal control. B, MEF cell sensitivities to low LET (L) or high LET (H) radiation at different doses as labeled were examined using a clonogenic assay. Data are the mean and S.D. (error bars) obtained from three independent experiments. **, p < 0.01. C, Ogg1 levels were detected by immunoblotting at 30 h after transfecting the Ogg1-containing vector or control vector into the MEF Ogg1−/− cells. D, the sensitivities of the MEF cells that transfected with either control vector without Ogg1 (Ogg1−/−) or vector encoding Ogg1 (+Ogg1) to low LET or high LET radiation at different doses, as labeled, were examined using a clonogenic assay. Data are the mean and S.D. obtained from three independent experiments. **, p < 0.01. E, the Ape1 levels were detected by Western blot at 30 h after transfecting the vector control, vector encoding Ape1 to the human cells (WT MRC5SV), control RNA, or siRNA against Ogg1 shown in the left frame. The Ogg1 levels are shown in the right frame. β-Actin was used as the internal control. F, the sensitivities of human cell MRC5SV (WT) or 180BRM (NHEJd; NHEJ-deficient) to low LET or high LET at different doses as labeled were examined using a clonogenic assay. Data are the mean and S.D. obtained from three independent experiments. **, p < 0.01. G, the RBE of NHEJd cells (M, Ku80−/− MEF cells; H, transformed 180BRM human fibroblast cells) in cell killing with or without Ape1 overexpression.

Article Snippet: The wild type, enzymatically overactivated or inactivated mouse Ape1 was amplified by PCR using plasmid pCMV6-kan/neo mAPEX1 (purchased from Origene Inc.) as template with proper primers ( ).

Techniques: Western Blot, Plasmid Preparation, Labeling, Clonogenic Assay, Transfection, Transformation Assay, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing *

doi: 10.1074/jbc.M114.604959

Figure Lengend Snippet: Up-regulation of Ape1 results in more unrepaired DNA DSBs in high LET- than in low LET-irradiated cells. A, left, image of γ-H2AX foci in wild type cells at 1 h after 1 Gy of high or low LET radiation; right, percentage of γ-H2AX foci-positive WT MEF cells transfected with control plasmid (CP) or the plasmid encoding Ape1 (AP) at different times (1, 4, and 24 h) after 1 Gy of high LET or low LET IR. B, NHEJ-deficient (NHEJd) (Ku80−/−) MEF cells in the same assay as described in A. C, percentage of γ-H2AX foci-positive Ogg1−/− MEF transfected with control vector or Ogg1−/− MEF cells with Ogg1 re-expressed (Ogg1+) (with or without overexpression of Ape1, as described in A, at different times (1, 4, and 24 h) after 1 Gy of high or low LET IR. Each point represents the mean ± S.D. (error bars) from two separate experiments (examining 300 cells for each experiment). *, p < 0.05. D, quantitation of γ-H2AX foci-positive cells in WT human MRC5SV cells transfected with control plasmid or the plasmid encoding Ape1 at different times (1, 4, and 24 h) after 1 Gy of high or low LET IR. Each point represents the mean ± S.D. from two separate experiments (300 cells examined during each experiment). *, p < 0.05. E, quantitation of γ-H2AX foci-positive cells in NHEJ-deficient human 180BRM cells transfected with control plasmid or the plasmid encoding Ape1 at different times (1, 4, and 24 h) after 1 Gy of high or low LET IR. Each point represents the mean ± S.D. from two separate experiments (300 cells examined during each experiment). *, p < 0.05.

Article Snippet: The wild type, enzymatically overactivated or inactivated mouse Ape1 was amplified by PCR using plasmid pCMV6-kan/neo mAPEX1 (purchased from Origene Inc.) as template with proper primers ( ).

Techniques: Irradiation, Transfection, Plasmid Preparation, Over Expression, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing *

doi: 10.1074/jbc.M114.604959

Figure Lengend Snippet: Up-regulation of Ape1 results in more Mre11 bound to chromatin DNA in high LET-irradiated cells. A, the levels of Mre11 or γ-H2AX associated with chromatin (Chr bound) were examined in the WT MEF cells transfected with control plasmid (Control) or the plasmid encoding Ape1 at 1 h after exposure to 10 Gy of low or high LET IR. The levels of Mre11 or H2A in whole cell lysates (WC lysates) were used as a loading control. The data represent the mean ± S.D. (error bars) from three independent experiments; *, p < 0.05. B, the levels of Mre11 or γ-H2AX associated with chromatin were examined in NHEJd (Ku80−/−) MEF cells transfected with control plasmid or the plasmid encoding Ape1 at 1 h after exposure to 10 Gy of low or high LET IR. The data represent the mean ± S.D. from three independent experiments; *, p < 0.05. C, the levels of Mre11 or γ-H2AX associated with chromatin were examined in Ogg1−/− MEF cells transfected with control plasmid or the plasmid encoding Ape1 at 1 h after exposure to 10 Gy of low or high LET IR. The data represent the mean ± S.D. from three independent experiments. D, the levels of Mre11 or γ-H2AX associated with chromatin were examined in Ogg1+ cells (Ogg1−/− cells with Ogg1 re-expressed) transfected with control plasmid or the plasmid encoding Ape1 at 1 h after exposure to 10 Gy of low or high LET IR. The data represent the mean ± S.D. from three independent experiments. *, p < 0.05.

Article Snippet: The wild type, enzymatically overactivated or inactivated mouse Ape1 was amplified by PCR using plasmid pCMV6-kan/neo mAPEX1 (purchased from Origene Inc.) as template with proper primers ( ).

Techniques: Irradiation, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing *

doi: 10.1074/jbc.M114.604959

Figure Lengend Snippet: Physiological levels of Ape1 contribute to high LET IR-induced RBE in cell killing. A, outline of the plasmid constructs of the enzymatically overactivated or inactivated mouse Ape1. B, description of the design for siRNA-resistant mouse Ape1 vector. The pink sites are mutated nucleotides. C, endogenous or exogenous Ape1 was detected by immunoblotting with the Ape1 or HA antibody in wild type MEF cells transfected with wild type (WT), inactivated (In), or overactivated (Ac) Ape1 combined with control (Ct) RNA or siRNA against Ape1 (siApe1). D, the relative Ape1 enzymatic activity in the MEF cells expressing different types of Ape1 was analyzed by comparing with the enzymatic activity of the cells transfected with wild type Ape1 when the endogenous Ape1 was knocked down without IR (NR). The cells were collected at 1 h after exposure to 4 Gy of low LET (L4 Gy) or 2 Gy of high LET (H2 Gy) radiation. The data were derived from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. E, the cell sensitivities to low LET (L) or high LET (H) irradiation at the indicated dose as labeled were examined using a clonogenic assay. Data are the mean and S.D. (error bars) obtained from three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The wild type, enzymatically overactivated or inactivated mouse Ape1 was amplified by PCR using plasmid pCMV6-kan/neo mAPEX1 (purchased from Origene Inc.) as template with proper primers ( ).

Techniques: Plasmid Preparation, Construct, Western Blot, Transfection, Activity Assay, Expressing, Derivative Assay, Irradiation, Labeling, Clonogenic Assay

Journal: The Journal of Biological Chemistry

Article Title: Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing *

doi: 10.1074/jbc.M114.604959

Figure Lengend Snippet: Small DNA DSB fragments (∼40 bp) do not affect HRR efficiency in vivo. A, HRR reporter plasmid design. pDR-GFP-40 was generated by introducing a second I-SceI digestion site 40 bp upstream from the original I-SceI site within the GFP coding region. B, the digestion efficiency of I-SceI in vitro. Top, efficiency of I-SceI digestion of the plasmid with one digestion site (1 dig site) or two digestion sites (2 dig sites) for I-SceI as described in A. The digestion efficiency was analyzed by PCR with proper primers. Bottom, based on the image of the gel, as shown in the top, we analyzed the in vitro digestion efficiency of I-SceI. Data represent the mean ± S.D. (error bars) from three independent experiments. **, p < 0.01. C, HRR efficiency was examined in human 293FT cells integrated with the substrate of I-SceI after transfecting with I-SceI plasmid. Data are the mean and S.D. obtained from three independent experiments. **, p < 0.01. D, a model explaining how high LET IR generates DSBs in mammalian cells. High LET radiation-induced small DNA fragments are generated from a direct energy transfer track to break DNA strands and from subsequent Ape1 enzymatic modification in the clustered damage DNA sites. These small DNA fragments interfere with Ku-dependent NHEJ but do not affect HRR.

Article Snippet: The wild type, enzymatically overactivated or inactivated mouse Ape1 was amplified by PCR using plasmid pCMV6-kan/neo mAPEX1 (purchased from Origene Inc.) as template with proper primers ( ).

Techniques: In Vivo, Plasmid Preparation, Generated, In Vitro, Modification

Journal: Disease Markers

Article Title: ZNF143 Suppresses Cell Apoptosis and Promotes Proliferation in Gastric Cancer via ROS/p53 Axis

doi: 10.1155/2020/5863178

Figure Lengend Snippet: ZNF143 inhibited cell cycle arrest in the G1 phase and the apoptosis of GC cells. (a, b) Cell cycle distribution of GC cells measured using flow cytometry. (c) Percentage of G1 phase. (d) Protein expression of CDC6, PLK1, MCM2, and MCM4 detected using Western blot analysis. (e, f) Apoptotic rate of GC cells measured using flow cytometry. (g) Apoptotic rate of GC cells. (h) Protein expression levels of p53, Bcl-2, Bax-cl, and Bax detected using Western blot analysis. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. The data were expressed as mean ± standard deviation. CDC6: cell division cycle 6; PLK1: polo-like kinase 1; MCM2: minichromosome maintenance complex component 2; MCM4: minichromosome maintenance complex component 4; p53: tumor protein 53; Bcl-2: B-cell lymphoma-2; Bcl-xl: BCL2-like 1; Bax: BCL2 associated X; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human wild-type p53 (RiboBio, Guangzhou, China) was transiently transfected into cells using Lipofectamine 2000 (Life Technologies, CA, USA) following the manufacturer's protocol.

Techniques: Flow Cytometry, Expressing, Western Blot, Standard Deviation

Journal: Disease Markers

Article Title: ZNF143 Suppresses Cell Apoptosis and Promotes Proliferation in Gastric Cancer via ROS/p53 Axis

doi: 10.1155/2020/5863178

Figure Lengend Snippet: Overexpression of ZNF143 inhibited p53-dependent ROS-mediated apoptosis in BGC823. (a) Protein level of ZNF143 detected using Western blot assay. (b) Apoptotic rate of GC cells measured using flow cytometry. (c, d) ROS level of GC cells detected using confocal microscopy. (e) Expression of ZNF143 and p53 in GC cells analyzed using Western blot assay. (f) Apoptotic rate of GC cells measured using flow cytometry. (g, h) ROS level of GC cells after different treatments were detected using confocal microscopy. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. The data were expressed as mean ± standard deviation. NAC: N-acetylcysteine; ROS, reactive oxygen species.

Article Snippet: Human wild-type p53 (RiboBio, Guangzhou, China) was transiently transfected into cells using Lipofectamine 2000 (Life Technologies, CA, USA) following the manufacturer's protocol.

Techniques: Over Expression, Western Blot, Flow Cytometry, Confocal Microscopy, Expressing, Standard Deviation

Journal: Disease Markers

Article Title: ZNF143 Suppresses Cell Apoptosis and Promotes Proliferation in Gastric Cancer via ROS/p53 Axis

doi: 10.1155/2020/5863178

Figure Lengend Snippet: Knockdown of ZNF143 could promote p53-dependent ROS-mediated apoptosis in HGC27. (a) Protein level of ZNF143 detected using Western blot analysis. (b) Apoptotic rate of GC cells measured using flow cytometry. (c, d) ROS level of GC cells detected using confocal microscopy. (e) Expression of ZNF143 and p53 in GC cells analyzed using Western blot assay. (f) Apoptotic rate of GC cells measured using flow cytometry. (g, h) ROS level of GC cells after different treatments were detected using confocal microscopy. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. The data were expressed as mean ± standard deviation.

Article Snippet: Human wild-type p53 (RiboBio, Guangzhou, China) was transiently transfected into cells using Lipofectamine 2000 (Life Technologies, CA, USA) following the manufacturer's protocol.

Techniques: Knockdown, Western Blot, Flow Cytometry, Confocal Microscopy, Expressing, Standard Deviation

Journal: Disease Markers

Article Title: ZNF143 Suppresses Cell Apoptosis and Promotes Proliferation in Gastric Cancer via ROS/p53 Axis

doi: 10.1155/2020/5863178

Figure Lengend Snippet: ZNF143 promoted xenograft tumor growth of GC cells in nude mice. (a, b) Tumors were obtained from nude mice injected subcutaneously with GC cells. (c) Growth curve of tumor volumes. (d) Average tumor weight of nude mice. (e) Representative images of ZNF143, p53, Ki67, and TUNEL staining in the xenografts. Original magnification, 200x. (f) Percentages of cells positive for Ki67 staining presented as a Ki67 index. (g) Percentages of TUNEL-positive cells presented as an apoptosis index. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. The data were expressed as mean ± standard deviation. Ki67: nuclear-associated antigen; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay.

Article Snippet: Human wild-type p53 (RiboBio, Guangzhou, China) was transiently transfected into cells using Lipofectamine 2000 (Life Technologies, CA, USA) following the manufacturer's protocol.

Techniques: Injection, TUNEL Assay, Staining, Standard Deviation, End Labeling

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Photograph of the cut section of an ovarian sclerosing stromal tumor (SST; left) displaying classic SST appearance with yellow tissue at periphery and white, central fibrotic depression, and micrographs of hematoxylin & eosin stained representative section at low (top right) and high (bottom right) magnification. Scale bars, 1 cm (left), 200 μm (top right), 50 μm (bottom right). b Schematic representation of the FHL2-GLI2 fusion transcript including the exons and domains involved. The breakpoint of the 5′ and 3′ partner genes are represented as black vertical lines. Spanning reads are depicted and aligned to the predicted junction sequence. c Schematic representation showing the Reads Per Kilobase per Million (RPKM) mapped read counts of each GLI2 exon. The GLI2 fusion breakpoint is represented as a red dashed line. d Fluorescence in situ hybridization (FISH) of two representative SSTs using a three-color FHL2-GLI2 probe, with 5′ GLI2 (orange), 3′ GLI2 (red), and 5′ FHL2 (green), showing the presence of the FHL2-GLI2 fusion (white arrows). e Representative Sanger sequencing electropherograms of the genomic FHL2-GLI2 breakpoint. f RNA in situ hybridization (RNA-ISH) using custom FHL2-GLI2 probes (red) showing the chimeric FHL2-GLI2 mRNA expression in two representative SSTs harboring the FHL2-GLI2 fusion. g Frequency of the FHL2-GLI2 fusion gene and GLI2 rearrangements in 26 SSTs from this study. h Frequency of the FHL2-GLI2 fusion gene and GLI2 rearrangements in 26 SSTs and frequency of the FHL2-GLI2 fusion gene in 48 other ovarian sex cord-stromal tumors from this study. aGCT, adult-type granulosa cell tumor. i Frequency of FHL2-GLI2 fusion gene in 26 SSTs from this study and in 9950 tumors from 33 cancer types from The Cancer Genome Atlas (TCGA). AML acute myeloid leukemia, PCPG pheochromocytoma and paraganglioma.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Staining, Sequencing, Fluorescence, In Situ Hybridization, RNA In Situ Hybridization, Expressing

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY), and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. b Representative images of colony formation assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 5 mm; top). Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2 or FHL2-GLI2. The migratory effects/wound area was assessed at 0 and 24 h (Scale bar, 500 μm; top) and quantified (bottom). In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Colony Assay, Wound Healing Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Quantitative assessment of CALB2 transcripts in immortalized mesenchymal stem cells (MSCs) and HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. b Representative western blot analysis of calretinin protein levels in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (bottom) of protein levels as compared to control. c Representative confocal micrographs of immunofluorescence analysis of calretinin (green) and 4–6-diamidino-2-phenylindole (DAPI, blue) in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 50 μm). Quantification (bottom) of calretinin intensity/cell relative to control. d Quantitative assessment of FOXL2 transcripts in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. In a – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Differential gene expression analysis of human sclerosing stromal tumors (SSTs) subjected to RNA-sequencing ( n = 8, this study) and high-grade serous ovarian carcinomas ( n = 16; The Cancer Genome Atlas) and other sex cord-stromal tumors (SCSTs, n = 11; this study). Gene expression fold-change is color-coded according to the legend. Only genes significantly differentially expressed ( P < 0.05; two-tailed unpaired t -test) are shown. CPM, count per million. b Expression levels of Sonic Hedgehog (SHH) pathway genes in human SSTs ( n = 11) and other sex-cord stromal tumors ( n = 9) as defined using NanoString. Expression levels and SHH enrichment scores are color-coded according to the legends. *** P < 0.001, Wilcoxon rank test. Hierarchical clustering was performed using complete linkage and Euclidian distance. c Quantitative assessment of the Sonic Hedgehog pathway PTCH1 and GLI1 transcripts in immortalized mesenchymal stem cells (MSCs), HEK-293 and medulloblastoma (DAOY) cells and of PTCH1 and CCND1 transcripts in human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. d Representative western blot analysis of PTCH1 and GLI1 protein expression in MSC, HEK-293 and DAOY cells and of PTCH1 and CCND1 in BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (below) of protein levels as compared to control. In c – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Gene Expression, RNA Sequencing, Two Tailed Test, Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Representative confocal micrographs of immunofluorescence analysis of FLAG (red), 4–6-diamidino-2-phenylindole (DAPI, blue), and GFP (green) in HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Scale bars, 10 μm. b GLI response element (GLI-RE) luciferase reporter assay of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (top), and GLI-RE promoter activity in HEK-293 transiently transfected with control, FHL2-GLI2 and FHL2-GLI2 with R338A-K339A mutations in the Zinc Finger 5 of GLI2 required for DNA binding (FHL2-GLI2 Mutated; bottom). SV40-Renilla was used to normalize transfection efficiency. c Immunoprecipitation assay with SUFU antibody of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Western blot analysis using anti-FLAG and anti-SUFU antibodies, and tubulin as loading control (left). GLI-RE promoter activity in HEK-293 stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 transfected with SUFU or control (right). d Cell titer blue proliferation assay of HEK-293, DAOY and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 20 µM GANT61 or vehicle control (DMSO). GANT, GANT61. e FLAG Chromatin Immunoprecipitation (ChIP) assay of GLI1 and PTCH1 promoters (promoter 1 and 2) in MSC and HEK-293 cells stably expressing either control or FHL2-GLI2. GLI1 and PTCH1 gene body and MYOD1, gene promoters not under GLI regulation, were used as negative controls. In b – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; *P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Reporter Assay, Activity Assay, Transfection, Binding Assay, Immunoprecipitation, Western Blot, Proliferation Assay, Chromatin Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY) and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2) or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). b Representative images of colony formation assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with Vismodegib 500 nM or vehicle control (DMSO). Scale bars, 5 mm. Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). The migratory effect/wound area was assessed at 0 and 24 h and quantified compared to DMSO (bottom). Vismo, Vismodegib. Scale bars, 500 μm. In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Colony Assay, Wound Healing Assay, Two Tailed Test