Journal: The Journal of Clinical Investigation

Article Title: Mutant SF3B1 promotes AKT- and NF- κ B–driven mammary tumorigenesis

doi: 10.1172/JCI138315

Figure Lengend Snippet: (A) GSEA identifies enrichment of hallmark gene sets of TNF-α signaling via NF-κB (top) and inflammatory response (bottom) in SF3B1 mutant versus WT human breast cell lines. FDR, false discovery rate; NES, normalized enrichment score. (B) Activation of phospho-p65 (P-p65) upon TNF-α stimulation of MCF10A SF3B1K700E/WT mutant knockin cells (top) and MCF7 with transgenic expression of SF3B1K700E versus SF3B1WT cDNA (bottom). Quantitation of 3 independent experiments is shown in Supplemental Figure 7B. (C) Levels of TGF-α, IL-1a, and IL-8 in culture media from MCF10A cells shown in B or IL-8 in culture media from MCF7 cells with expression of an empty vector (EV), SF3B1 WT, or SF3B1 K700E cDNAs measured by Milliplex cytokine/chemokine multiplex assay. (D) Levels of IL-8 in culture media shown in C measured by ELISA. Mean levels with standard deviation shown. (*P < 0.05, **P < 0.01, ***P < 0.0001; Student’s t test for MCF10A and Dunnett’s multiple comparisons test for MCF7). (E) Venn diagrams of genes significantly differentially expressed (P < 0.05; DESeq2) and spliced (q < 0.05; t test) across SF3B1 mutant versus WT human breast cancers from TCGA (22) (left), isogenic human breast cell lines (right), and overlapping across human tumors, human cell lines, and mouse mammary epithelial cells mutant for Sf3b1 (middle). See also Supplemental Figure 7.

Article Snippet: MCF7 and MCF10A cells were obtained from ATCC.

Techniques: Mutagenesis, Activation Assay, Knock-In, Transgenic Assay, Expressing, Quantitation Assay, Plasmid Preparation, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: The Journal of Clinical Investigation

Article Title: Mutant SF3B1 promotes AKT- and NF- κ B–driven mammary tumorigenesis

doi: 10.1172/JCI138315

Figure Lengend Snippet: Representative RNA-seq coverage plots showing cryptic 3′ss usage in MAP3K7 in (A) human isogenic breast cancer cell lines (MCF10A, MCF7, T47D) expressing WT or SF3B1 K700E (K700E) and human breast tumor samples from TCGA (22) as well as (B) mouse mammary epithelial cells from MMTV-cre control, MMTV-cre Sf3b1K700E/WT, MMTV-cre R26-LSL-Pik3caH1047R transgenic, and compound MMTV-cre Sf3b1K700E/WT R26-LSL-Pik3caH1047R mice. PSI value is shown on the right of each coverage plot. (C) MAP3K7 mRNA expression (normalized log2RPKM values) in SF3B1 mutant (MUT) versus WT human breast tumors from TCGA and cell lines shown in A. Box indicates upper and lower quartiles; thick bar indicates the median value. Whiskers indicate the largest/smallest value no further than 1.5 times the interquartile range. (D) RT-PCR of MAP3K7 splicing event (top) and MAP3K7 protein levels in isogenic MCF10A cells with or without SF3B1K700E knockin. The 3’ss used is shown in the schematic. (E) MAP3K7 protein levels in MCF7 cells expressing doxycycline-inducible FLAG-SF3B1WT or FLAG-SF3B1K700E vectors. RT-PCR of MAP3K7 splicing is shown on bottom. (F) RT-PCR of MAP3K7 splicing event derived from MAP3K7 minigene with mutations at aberrant branchpoint or polypyrimidine region downstream of the aberrant 3’ss. RT-PCR of MAP3K7 splicing event derived from endogenous MAP3K7 is shown on bottom. A schematic of sequences around the normal and aberrant branchpoint and splice site is shown on top. (G) MAP3K7 and phospho- and total p65 and p38 levels in MCF7 and MCF10A cells transduced with anti-MAP3K7 shRNA. (H) MAP3K7 and phospho- and total p65 and p38 levels in MCF7 and MCF10A cells expressing SF3B1 K700E mutant from D and E transduced with or without MAP3K7-GFP cDNA. Quantitation of 3 independent experiments is shown in Supplemental Figure 8. See also Supplemental Figure 9.

Article Snippet: MCF7 and MCF10A cells were obtained from ATCC.

Techniques: RNA Sequencing, Expressing, Control, Transgenic Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Knock-In, Derivative Assay, Transduction, shRNA, Quantitation Assay

Journal: The Journal of Clinical Investigation

Article Title: Mutant SF3B1 promotes AKT- and NF- κ B–driven mammary tumorigenesis

doi: 10.1172/JCI138315

Figure Lengend Snippet: Representative RNA-seq coverage plots showing cryptic 3′ss usage in PPP2R5A in (A) human isogenic breast cancer cell lines (MCF10A, MCF7, T47D) expressing WT or SF3B1 K700E (K700E) and human breast tumor samples from TCGA (22) as well as (B) mouse mammary epithelial cells from MMTV-cre control, MMTV-cre Sf3b1K700E/WT, MMTV-cre R26-LSL-Pik3caH1047R transgenic, and compound MMTV-cre Sf3b1K700E/WT R26-LSL-Pik3caH1047R mice. PSI value of each event is shown on the right of each coverage plot. (C) PPP2R5A mRNA expression (normalized log2RPKM values) in SF3B1 mutant (MUT) versus WT primary human breast tumors from TCGA and cell lines shown in A. Box indicates upper and lower quartiles; thick bar indicates the median value. Whiskers indicate the largest/smallest value no further than 1.5 times the interquartile range. (D) PPP2R5A, phospho- and total AKT, and p65 levels in MCF7 and MCF10A with and without mutant SF3B1 expression (EV, empty vector). Quantitation of 3 independent experiments is shown in Supplemental Figure 10A. (E) RT-PCR of PPP2R5A splicing event derived from PPP2R5A minigene with mutations at aberrant branchpoint or polypyrimidine region downstream of the aberrant 3′ss. RT-PCR of PPP2R5A splicing event derived from endogenous PPP2R5A is shown on the bottom. A schematic of sequences around the normal and aberrant branchpoint and splice site is shown on top. (F) PPP2R5A, phospho- and total AKT, and p65 levels in MCF7 and MCF10A with and without PPP2R5A shRNA knockdown. Quantitation of 3 independent experiments is shown in Supplemental Figure 10B. (G) Relative mRNA expression of IL1A, CXCL1, IL8, CDKN1A, and PPP2R5A in the cells from D and F. Mean ± standard deviation shown; asterisks represent P values of Dunnett’s test compared with shRenilla control (shRE). See also Supplemental Figures 9 and 10.

Article Snippet: MCF7 and MCF10A cells were obtained from ATCC.

Techniques: RNA Sequencing, Expressing, Control, Transgenic Assay, Mutagenesis, Plasmid Preparation, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, shRNA, Knockdown, Standard Deviation

Journal: The Journal of Clinical Investigation

Article Title: Mutant SF3B1 promotes AKT- and NF- κ B–driven mammary tumorigenesis

doi: 10.1172/JCI138315

Figure Lengend Snippet: (A) Representative photos (top) at 0, 24, and 48 hours following wound creation in cell culture of MCF7 cells expressing an empty vector (EV), SF3B1WT, or SF3B1K700E cDNA. Scale bars: 200 μM. Relative scratch width over time shown below (n = 3; mean value ± standard deviation shown below; P values by ANOVA). (B) Quantification and representative photos (left) of wound at 0, 8, and 24 hours following wound creation and exposure to 0, 2, and 10 ng/mL TNF-α in cell culture of MCF10A with or without knockin of SF3B1K700E. Right, mean levels with standard deviation shown (n = 3; P value by Student’s test). Scale bars: 500 μM. (C) Same as A but for parental MCF7 cells exposed to medium from cells in A. Scale bars: 200 μM. (D) Number of invaded MCF10A cells with or without knockin of SF3B1K700E migrating toward EGF in a Transwell invasion assay (left). Mean ± standard deviation (n = 3, P value by Student’s t test) shown. Right, photos of hematoxylin staining of the migrated cells (photos taken using a microscope with a 10× objective (total magnification ×100); scale bars: 200 μM). (E) Growth of MCF7 tumors expressing either WT or mutant SF3B1 in NSG mice. Size of individual tumor (n = 10 in each group) and the mean are shown (P value by ANOVA). See also Supplemental Figures 11 and 12.

Article Snippet: MCF7 and MCF10A cells were obtained from ATCC.

Techniques: Cell Culture, Expressing, Plasmid Preparation, Standard Deviation, Knock-In, Transwell Invasion Assay, Staining, Microscopy, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Mutant SF3B1 promotes AKT- and NF- κ B–driven mammary tumorigenesis

doi: 10.1172/JCI138315

Figure Lengend Snippet: (A) Western blot of MCF7 cells expressing an empty vector (EV), SF3B1 WT, or SF3B1 K700E cDNA. (B) IC50 of MCF7 cells shown in A following 5 days of exposure to MK2206 (left) or GDC0068 (right). Data are represented as mean ± SD; n = 6. (C) Western blot of MCF10A WT cells as well as SF3B1K700E/WT knockin cells, PIK3CAH1047R/WT knockin cells, and SF3B1K700E/WT PIK3CAH1047R/WT double knockin cells cultured in the absence of EGF. (D) IC50 to MK2206 of MCF10A cells shown in C in the presence or absence of EGF. Data are represented as mean ± SD; n = 6. (E) Clonogenic survival assay of MCF7 cells shown in A following 21 days of exposure to MK2206. IC50 curve is shown at right. (F) Clonogenic survival assay of MCF710A cells shown in C following 14 days of exposure to MK2206 in absence of EGF. IC50 curve is shown at right. See also Supplemental Figures 13 and 14.

Article Snippet: MCF7 and MCF10A cells were obtained from ATCC.

Techniques: Western Blot, Expressing, Plasmid Preparation, Knock-In, Cell Culture, Clonogenic Cell Survival 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 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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Targeting SGK1 enhances the efficacy of radiotherapy in locally advanced rectal cancer.

doi: 10.1016/j.biopha.2020.109954

Figure Lengend Snippet: Fig. 2. (A) Representative images of the SGK1 expression in pCR (top,radiosensitive group) and non-pCR (bottom, radioresistance group); (B) SGK1 mRNA expression (left) and the cell survival curves (right) were measured in the HT29, RKO, LoVo, and HCT116 cell lines. (C) Western blot and quantitative real time polymerase chain reaction (qRT-PCR) showing the SGK1 expression compared with the control cells in the LoVo, HCT116, and HT29 cells. Data are reported as relative quantity ± SD with statistical significance at* p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The human colorectal cancer cell lines LoVo, HT29, and HCT116 were obtained from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Targeting SGK1 enhances the efficacy of radiotherapy in locally advanced rectal cancer.

doi: 10.1016/j.biopha.2020.109954

Figure Lengend Snippet: Fig. 3. SGK1 expression altered the radioresistance of the colorectal cancer cells. (A) Clonogenic cell survival of the LoVo and HCT116 cells stably transfected with the SGK1 or control. (B) Representative image of clonogenetic survival in the LoVo and HCT116 cells stably transfected with SGK1, or the control, and exposed to radiation therapy (RT). (C) (left)Clonogenic cell survival curves of the RKO cells stably transfected with SGK1(KM) or SGK1; (right)representative image of the clonogenetic survival assay of the RKO cells stably transfected with SGK1(KM) or SGK1. The surviving fraction is reported as the mean ± SD values from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The human colorectal cancer cell lines LoVo, HT29, and HCT116 were obtained from the American Type Culture Collection.

Techniques: Expressing, Stable Transfection, Transfection, Control, Clonogenic Cell Survival Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Targeting SGK1 enhances the efficacy of radiotherapy in locally advanced rectal cancer.

doi: 10.1016/j.biopha.2020.109954

Figure Lengend Snippet: Fig. 4. The pharmacologic inhibition of SGK1 reduced the radioresistance of colorectal cancer in vitro. (A) The clonogenic cell survival of LoVo and HT29 cells treated with SGK1 inhibitor or the control. The surviving fraction is reported as mean ± SD values from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001. (B) Representative image of the clonogenetic survival assay in LoVo and HT29 cells stably treatment with a SGK1 inhibitor, or the control exposed to RT.

Article Snippet: The human colorectal cancer cell lines LoVo, HT29, and HCT116 were obtained from the American Type Culture Collection.

Techniques: Inhibition, In Vitro, Control, Clonogenic Cell Survival Assay, Stable Transfection

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Targeting SGK1 enhances the efficacy of radiotherapy in locally advanced rectal cancer.

doi: 10.1016/j.biopha.2020.109954

Figure Lengend Snippet: Fig. 5. The pharmacologic inhibition of SGK1 reduced the radioresistance of colorectal cancer in vivo. (A) Landscape of pharmacologic inhibition in vivo. (B) Weight of tumors treated with DMSO(negative control, NC), RT, and SGK1 inhibitor, or with RT combined with an SGK1 inhibitor. (C) Image of a tumor treated with a DMSO, RT, and SGK1 inhibitor, or with RT combined with an SGK1 inhibitor. (D) Growth curves of the growth of the HT29 xenographs treated with DMSO (black, n = 10), RT (red, n = 10), and SGK1 inhibitor (green, n = 10), or RT combined with SGK1 inhibitor (blue, n = 11). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The human colorectal cancer cell lines LoVo, HT29, and HCT116 were obtained from the American Type Culture Collection.

Techniques: Inhibition, In Vivo, Negative Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Targeting SGK1 enhances the efficacy of radiotherapy in locally advanced rectal cancer.

doi: 10.1016/j.biopha.2020.109954

Figure Lengend Snippet: Fig. 6. Gene expression profiling identified an association between SGK1 alterations and activate transcription factor 3 (ATF3). (A) Differential gene expression profiling of LoVo with the pharmacologic inhibition of SGK1, or RT combined with an SGK1 inhibitor, by transcriptome analysis. (B) Difference analysis of ATF3 of LoVo cells with the pharmacologic inhibition of SGK1, or RT combined with an SGK1 inhibitor (C) qRT-PCR analysis of the ATF3 expression in LoVo cells with the pharmacologic inhibition of SGK1, or RT combined with an SGK1 inhibitor. The analysis is reported as mean ± SD values from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The human colorectal cancer cell lines LoVo, HT29, and HCT116 were obtained from the American Type Culture Collection.

Techniques: Gene Expression, Inhibition, Quantitative RT-PCR, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Targeting SGK1 enhances the efficacy of radiotherapy in locally advanced rectal cancer.

doi: 10.1016/j.biopha.2020.109954

Figure Lengend Snippet: Fig. 7. The ATF3 expression reduced the radioresistance of colorectal cancer cells. (A) qRT-PCR showing the ATF3 expression compared to control cells in HCT116 (left); clonogenic cell survival curves of HCT116 cells stably transfected with ATF3 or the control (right). (B) qRT-PCR showing the ATF3 expression compared with the control cells in the HCT116 cells stably transfected with ATF3 short hairpin RNA (shRNA) or control (left); clonogenic cell survival curves of HCT116 cells stably transfected with ATF3 shRNA or the control (right). The surviving fraction is reported as mean ± SD values from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The human colorectal cancer cell lines LoVo, HT29, and HCT116 were obtained from the American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Control, Stable Transfection, Transfection, shRNA

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) Growth curves of wild-type Salmonella and its isogenic sifA mutant in LB media in the presence of pEVOL. The error bars represent the mean ± standard deviation (n = 3). ( B ) Survival assay of wild-type Salmonella and wild-type Salmonella transformed with pEVOL in HeLa cells. Strains of Salmonella wild-type and the wild-type transformant with pEVOL were grown in LB to late exponential phase and added to HeLa cells at an MOI of 100:1 for 30 min. At 2 hr and 16 hr post infection, cells were lysed, and the intracellular bacteria were enumerated. The percentage of survival of the transformant was normalized to wildtype (100%). Figure 3—figure supplement 1—source data 1. Source data for . Figure 3—figure supplement 1—source data 2. Source data for .

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Mutagenesis, Standard Deviation, Clonogenic Cell Survival Assay, Transformation Assay, Infection, Bacteria

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with a sifA null strain of Salmonella expressing SifA-E52AzF and incubated for 16 hr. Infected cells were fixed with 4% PFA and immuno-stained for Salmonella (yellow) and LAMP1 (red). Images were acquired by spinning-disk SIM. Cells infected with sifA harboring p sifA 52TAG bacteria were capable of forming SIFs that resembled those of wild-type infected cells. ( B ) SPAAC labeling of secreted SifA-E52AzF with BDP-DBCO. HeLa cells were infected with a sifA null strain expressing SifA-E52AzF in the presence of 1 mM AzF. AzF-tagged secreted SifA-E52AzF was labeled with BDP-DBCO (green). Host nuclei and Salmonella nucleoids were labeled with DAPI (blue) and images were acquired using spinning-disk SIM. ( C ) SifA-E52AzF labeling was absent in the absence of ncAA. HeLa cells were infected with the sifA null mutant of Salmonella expressing SifA-E52AzF in the absence of AzF for 12 hr. After 12 hr post infection, HeLa cells were incubated with 2.5 μM BDP-DBCO in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive washing to remove excess dye as described in Methods. At 16 hr post-infection, cells were fixed. SifA was absent in the infected cells that lacked AzF. Images were acquired by confocal microscopy. See for statistical analysis for the average fraction of infected cells that contain LAMP1 positive SIFs and SIFs containing labeled SifA. ( D ) Colocalization of SifA with LAMP1. Salmonella secreted SifA-E52AzF in HeLa cells was labeled with BDP-DBCO (green). Cells were also immuno-stained for the endosomal membrane marker LAMP1 (red, middle panel). Images were acquired by confocal microscopy. From the merged image (right), it is evident that SifA is present within the SIFs. Statistical analysis is shown in . The data are representative of at least three independent experiments. Scale bar, 10 µm ( A–D ).

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Expressing, Incubation, Staining, Bacteria, Labeling, Mutagenesis, Confocal Microscopy, Membrane, Marker

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: HeLa cells were infected with Salmonella for 16 hr, fixed and immuno-stained for LAMP1 (red), LPS (yellow), and DAPI (blue). As expected, the formation of SIFs was observed in cells infected with WT Salmonella ( A ), but not in cells infected with the sifA null strain ( B ). Images were acquired on a spinning-disk SIM, as described in Materials and methods (n = >10 independent experiments). Scale bar, 10 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Staining

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( Ai ) SifA colocalizes with SseJ. Spinning-disk SIM image of an infected HeLa cell, 16 hr after invasion, fixed with PFA and stained for SifA (green) and SseJ-HA (red). ( Aii ) Magnified view of SifA and SseJ in the boxed region at the far left (arrows indicate colocalization). ( iii ) Colocalization was analyzed by calculating Mander’s M1 coefficient (SifA colocalizing with SseJ) for the original dual-color image (x = y = 0) and for control images generated by translating the spectral channels with respect to each other in x and y directions. This analysis generated a colocalization map, with a peak intensity at x = y = 0, indicating true colocalization (n = 3 cells). ( iv ) Colocalization analysis of mock-infected cells (n = 3 cells) stained for SifA-E52AzF and SseJ-HA showed no peak intensity at x = y = 0, indicating no colocalization of anti-HA antibodies and SifA. Bars, 10 µm ( i ), 5 µm (ii). ( B ) SifA colocalizes with kinesin. ( i ) Spinning-disk SIM image of an infected HeLa cell fixed with PFA at 16 hr post-infection and stained for kinesin (red) and SifA (green). (ii) Magnified view of SifA and kinesin in the boxed region (arrows indicate colocalization). (iii and iv) Colocalization analysis was performed as described in ( A ). The same color map scaling was used for comparison. px, pixels. See for statistical analysis. Scale bar, 5 µm ( i ), 2 µm (ii). ( C ) Bioorthogonal fluorescence imaging of SseJ-Y10TCO-HA with tetrazine fluorophores versus immunofluorescence staining. HeLa cells expressing SseJ-Y10TCO-HA in the presence of TCO*Lys were labeled with BDP-Tz under physiological conditions, briefly washed, and subjected to anti-HA immunofluorescence staining. Images were acquired by confocal microscopy. The data are representative of at least three independent experiments. Scale bar, 10 µm. Figure 4—source data 1. Source data for .. Figure 4—source data 2. Source data for .

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Staining, Control, Generated, Comparison, Fluorescence, Imaging, Immunofluorescence, Expressing, Labeling, Confocal Microscopy

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: HeLa cells were infected with WT -Salmonella harboring SseJ-HA and pEVOL in the presence of AzF. At 10 hr post infection, excess AzF was washed out and replaced with fresh growth media without AzF for 2 hr. At 14 hr post infection, HeLa cells were incubated with 2.5 μM BDP-DBCO in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive removal of excess dye with fresh growth media as described in Materials and methods. At 16 hr post-infection, cells were fixed and washed with PBS, then immunostained for SseJ (red). The absence of any noticeable fluorescence signal in the green channel (BDP-DBCO) indicated that there was virtually no off-target labeling of secreted effectors within host cells. The results presented in each panel were obtained in at least three independent experiments. Scale bar, 10 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Incubation, Fluorescence, Labeling

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with sifA mutants harboring p SifA 52TAG and pEVOL-AzF. At 10 hr post infection, excess AzF was washed out and replaced with fresh growth media without AzF for 2 hr. At 14 hr post infection, HeLa cells were incubated with 2.5 μM BDP-DBCO in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive removal of excess dye with fresh growth media as described in Materials and methods. At 16 hr post-infection, cells were extracted for 1 min using a microtubule-stabilizing buffer (MTSB). The buffer was exchanged with MTSB containing 0.2% GA, and cells were fixed for 10 min at room temperature. Excess GA was quenched for 7 min using 0.2% sodium borohydride in PBS. After several washes with PBS, cells were immunostained for microtubules (green). Scale bar, 10 µm. ( B ) Prior to immunostaining, GA-fixed HeLa cells were imaged for SifA labeled with BDP-DBCO using Spinning-disk SIM. Note the punctate labeling of SifA, indicating that while SseJ forms continuous filaments, SifA does not. The results presented in each panel were obtained in at least three independent experiments. Scale bar, 5 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Incubation, Immunostaining, Labeling

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with the sifA null strain of Salmonella expressing SifA-E52Hco and SseJ-HA for 16 hr, fixed and immuno-stained for LAMP1 (green), SseJ (red). The SifA-E52Hco strain was capable of forming SIFs and colocalized with SseJ. Images were acquired on a spinning-disk SIM as described in Materials and methods. ( B ) Direct visualization of secreted SifA. HeLa cells were infected with a sifA null strain of Salmonella expressing SifA-E52Hco in the presence of 1 mM Hco and fixed 16 hr post infection with 4% PFA. Hco-tagged secreted SifA-E52Hco was directly imaged by two photon microscopy. Immuno-stained SseJ-HA was also imaged in the same cell. The data are representative of at least three independent experiments. Scale bar, 10 µm ( A ), 20 µm ( B ).

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Expressing, Staining, Microscopy

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with the ssaP null mutant for 16 hr, fixed and immuno-stained for LPS (green), SseJ (red) and DAPI (blue). In the absence of ssaP , SseJ is not secreted. ( B ) TCO*Lys-incorporated SsaP was functional. HeLa cells were infected with Salmonella complemented with p ssaP 65TAG- sseJ -HA, fixed and immunostained for LPS (red), LAMP1 (green) and DAPI (blue). SIF formation was observed in cells infected with Salmonella complemented with p ssaP 65TAG- sseJ -HA. ( C ) Engineered SsaP rescued SPI-2 secretion. HeLa cells were infected with Salmonella complemented with p ssaP 65TAG- sseJ -HA, fixed and immunostained for LPS (Green), SseJ (red), and DAPI (blue). SseJ (red) was secreted in the complemented strain, but not in the ssaP null strain (n = >10, independent experiments). Scale bar, 10 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Mutagenesis, Staining, Functional Assay

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: Strains of Salmonella wild-type, ssaP null, ssaP/ λ ssaP (complemented with ssaP integrated a λ att ), and ssaP/ λ ssaP-55HA ( ssaP with an HA tag at amino acid 55) were grown in LB to late exponential phase and added to HeLa cells at an MOI of 100:1 for 30 min. At 2 hr and 14 hr, cells were lysed and the intracellular bacteria were enumerated. The normalized fold changes were relative to those of the wildtype (set to 1). The error bars represent the mean ± standard deviation (n = 3).

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Bacteria, Standard Deviation

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: Strains of Salmonella containing ssaP/λssaP-55HA (left) ssaP/ λssaP-110HA (middle and right) were grown in LB to late exponential phase and added to HeLa cells at an MOI of 100:1 for 30 min. The cells were fixed and immuno-stained for SsaP (red). A representative image at 14 hr post infection is shown. DAPI-stained DNA is in blue and GFP-expressing Salmonella are shown in green. The middle and right panels are of SsaP at different z positions.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Staining, Infection, Expressing

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) SPIEDAC labeling of secreted SifA-Y65TCO with fluorogenic SiR-Tz. HeLa cells were infected with the ssaP null mutant of Salmonella expressing SsaP-Y65TCO in the presence of TCO*Lys for 12 hr. After 12 hr post infection, HeLa cells were incubated with 1.5 μM SiR-Tz in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive wash out of excess dye with fresh growth media as described in Materials and methods. At 16 hr post-infection, cells were fixed. Cells were also immuno-stained for the endosomal membrane marker LAMP1 (green), LPS (Red) and DAPI (Blue) and imaged by spinning-disk SIM. From the merged image (right), it is evident that SsaP is present within the LAMP1-positive endosomes. A higher magnification spinning-disk SIM image of a SIF-positive HeLa cell from the boxed region ( i ) is shown on the right-most panel without the green channel displayed ( i ). It clearly shows secreted SsaP from Salmonella (boxed region). ( B ) SsaP was absent in infected cells that lacked ssaP . HeLa cells that were infected with an ssaP null mutant in the presence of TOC*Lys lack an SsaP signal. Note the absence of SIFs in the merged image (right) in the ssaP null strain. The data are representative of at least three independent experiments. Scale bar, 5 µm (A and i) and 10 µm ( B ).

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Labeling, Infection, Mutagenesis, Expressing, Incubation, Staining, Membrane, Marker

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with an ssaP null mutant of Salmonella expressing SsaP-YTCO*Lys in the presence of TCO*Lys. After 6 hr post infection, HeLa cells were incubated with 1.5 μM SiR-Tz in DMEM with 10% FBS for another 1 hr, followed by extenstive washing of excess dye with fresh growth media. At 8 hr post-infection, cells were fixed and immunostained for the endosomal membrane marker LAMP1 (green, left panel), LPS (red) and DAPI (blue). Scale bar, 5 µm. ( B ) Zoomed view of inset. Yellow arrow indicates polarly localized SsaP. Images were acquired on a spinning-disk SIM, as described in Materials and methods. Scale bar, 2 µm. ( C ) Not every pole where SsaP was located had an injectisome. Salmonella cells expressing SsaP-Y65TCO in the presence of TCO*Lys were labeled with SiR-Tz as described in Materials and methods. The same cells were immuno-stained for SseB using rabbit anti-SseB antibody (green) and imaged along with SiR fluorescence (magenta). The arrows indicate cells that have both SsaP and SseB. The data are representative of at least three independent experiments. Scale bar, 2 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Mutagenesis, Expressing, Incubation, Membrane, Marker, Labeling, Staining, Fluorescence

Journal: Pharmaceutics

Article Title: Impact of circ-0000221 in the Pathogenesis of Hepatocellular via Modulation of miR-661–PTPN11 mRNA Axis

doi: 10.3390/pharmaceutics14010138

Figure Lengend Snippet: Effect of hsa_circ_0000221 overexpression in HepG2 and SNU449 cells. hsa_circ_0000221 , miR-661, and PTPN 11 mRNA were quantified by qRT-PCR 1 and 2 days after transfection of either pcDNA-CIRC-0000221 or control vector in ( A ) HepG2 and ( B ) SNU449 cell lines. Data for each gene were normalized to their counterparts in cells at 0 h. ( C ) Cell count in HepG2 cells, and cellular viability in ( D ) HepG2 and ( E ) SNU449 cells were measured 1 and 2 days after transfection. Error bars represent SD. Statistics were performed using Student’s unpaired t -test; ** p ≤ 0.01, * 0.01 < p ≤ 0.05, and ns, p > 0.05.

Article Snippet: The human hepatocellular carcinoma cell lines used in this study, HepG2 and SNU449, were obtained from ATCC.

Techniques: Over Expression, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Cell Counting

Journal: Pharmaceutics

Article Title: Impact of circ-0000221 in the Pathogenesis of Hepatocellular via Modulation of miR-661–PTPN11 mRNA Axis

doi: 10.3390/pharmaceutics14010138

Figure Lengend Snippet: Exogenous expression of hsa-circ-0000221 induces apoptosis in HepG2 and SNU449 cells. ( A ) Phase contrast of SNU449 cells 1, 2, and 3 days after transient transfection with hsa-circ-0000221 -expressing vector or control. Clonogenic survival assay of ( B ) SNU449 and ( C ) HepG2 cells after overexpression of hsa-circ-0000221 or empty vector reflecting the differential colony-forming ability between the two conditions as indicated by the surviving fraction. Annexin-positive cell percentage as determined by flow cytometry of ( D ) SNU449 and ( E ) HepG2 cells after overexpression of hsa-circ-0000221 or empty vector following annexin V and propidium iodide staining. Statistics were performed using Student’s unpaired t -test; ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: The human hepatocellular carcinoma cell lines used in this study, HepG2 and SNU449, were obtained from ATCC.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Clonogenic Cell Survival Assay, Over Expression, Flow Cytometry, Staining

Journal: DNA repair

Article Title: XRCC1 and Base Excision Repair Balance in Response to Nitric Oxide

doi: 10.1016/j.dnarep.2011.10.008

Figure Lengend Snippet: A. XRCC1 WT (AA8) and null (EM9) CHO cells, as well as human XRCC1 complemented EM9 (H9T3-6-3 and H9T3-7-1) cells were used to assess the toxicity of (B) MMS, (C) the ONOO− donor SIN-1 and (D) gaseous NO·, using a clonogenic survival assay. P values were produced by a two-tailed t-test and only values below 0.05 are displayed.

Article Snippet: 2.1 Cells and Cell Culture AA8 and EM9 CHO cells were obtained from ATCC (Manassas, VA) and the human complemented EM9 cells were engineered, and kindly provided by the lab of Larry Thompson [ 46 ].

Techniques: Clonogenic Cell Survival Assay, Produced, Two Tailed Test

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet: ( A ) CtIP depletion and the expression of eGFP-tagged wild-type CtIP was monitored by western blot with antibody directed against CtIP (Active Motif, #61141). ( B ) CtIP depletion with shRNA was performed in U2OS cells and validated by western blot with antibody specific for human CtIP. The C-terminal domain of Senataxin (a.a. 1851–2677) with a V5 tag was induced from a tetracycline-induced promoter in a stably integrated transgene and verified with antibody directed against V5. ( C ) Cell viability assays were performed in U2OS cells with CtIP depletion and overexpression of Senataxin c-term or full-length PCF11 as indicated. * and *** denote p < 0.05 or 0.001, respectively, using Student's two-tailed T test with comparisons as indicated. ( D ) Global resection was monitored by western blot for phospho-S4/S8 RPA32 antibody in U2OS cells depleted of shCtIP and complemented with GFP-CtIP, PCF11, or C-term of Senataxin ( E ) Depletion of CtIP and expression of wild-type (wt), or nuclease deficient (NA/HA) CtIP was monitored by western blot with antibody directed against CtIP. ( F ) ( G ) Depletion of Senataxin, CtIP, and XPG were monitored by western blot with antibodies directed against CtIP, XPG (GeneTex, #GTX110609) and Senataxin (Novus Biologicals, #NB100-57542), respectively. ( H ) CtIP, XPG, or CtIP/XPG-depleted U2OS cells were exposed to increasing concentrations of CPT (1 hr). Cell viability was determined by clonogenic survival assay in comparison to untreated cells. Results are shown from three biological replicates and error bars indicate S.D.

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Expressing, Western Blot, shRNA, Stable Transfection, Over Expression, Two Tailed Test, Clonogenic Cell Survival Assay, Comparison

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet: ( A ) CtIP-depleted U2OS cells complemented with vector only or constructs overexpressing eGFP-CtIP, PCF11, or Senataxin (C-terminus) as indicated were exposed to increasing concentrations of CPT (1 hr). Cell viability was determined by clonogenic survival assay in comparison to untreated cells. Results are shown from three biological replicates and error bars represent S. D. ( B ) Wild-type or CtIP-depleted U2OS cells were pre-treated with DRB (20 μM) prior to CPT exposure and cell viability was determined as in ( A ). ( C ) Live cell imaging was performed with U2OS cells stably expressing RNaseH D10R-E48R -mCherry. The circle indicates the site of laser damage. ( D ) CtIP-depleted U2OS cells were complemented with eGFP-CtIP, PCF11, or Senataxin (C-terminus) as indicated and RNaseH D10R-E48R -mCherry accumulation at the laser damage was quantified. The average of 4 cells is shown and error bars represent S. E. M. ( E ) RNaseH D10R-E48R -mCherry accumulation at laser damage sites was measured in CtIP-depleted U2OS cells complemented with wild-type or nuclease deficient (‘NA/HA’) eGFP-CtIP as in ( D ); n > 12, error bars represent S.E.M. ( F ) Wild-type or CtIP-depleted U2OS cells were pre-treated with DRB (20 μM) prior to measurement of RNaseH D10R-E48R -mCherry accumulation at laser damage sites as in ( D ); n > 5; error bars represent S.E.M. ( G ) RNaseH D10R-E48R -mCherry accumulation at laser damage sites was measured in wild-type or CtIP-depleted, XPG-depleted, or Senataxin (SETX)-depleted U2OS cells as in ( D ); n = 5; error bars represent S.E.M. ( H ) RNaseH D10R-E48R -mCherry accumulation at laser damage sites was measured in wild-type, CtIP-depleted, XPG-depleted, or both CtIP/XPG-depleted U2OS cells as in ( D ); n > 8, error bars represent S.E.M. Results shown are representative of several experiments performed.

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Plasmid Preparation, Construct, Clonogenic Cell Survival Assay, Comparison, Live Cell Imaging, Stable Transfection, Expressing

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet: ( A ) Imaging of RNaseH D10R-E48R -mCherry or mCherry only was performed in U2OS cells with CtIP depletion as indicated. 7 and 3 cells were imaged for RNaseH D10R-E48R -mCherry and mCherry-only conditions, respectively. ( B ) Quantification of RNA/DNA hybrids using RNaseH D10R-E48R -mCherry or mCherry only, stably expressed in human U2OS cells by FACS in cells with CtIP depletion by shRNA. Cells were evaluated in G1, S, and G2 cell cycle phases as determined by DNA content (FxCycle Far Red Stain). ** and **** denote p < 0.01 or 0.0001, respectively, using Student's two-tailed T test with comparisons as indicated. ( C ) CtIP and XPG depletion with shRNA was performed in U2OS cells and validated by western blot with antibody specific for human CtIP or XPG, respectively. ( D ) CtIP depletion with shRNA and expression of wild-type or nuclease-deficient (NA/HA) mutant was performed in U2OS cells and validated by western blot with antibody specific for human CtIP. ( E ) CtIP and XPG depletion with shRNA was performed in U2OS cells and validated by western blot with antibody specific for human CtIP or XPG, respectively .

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Imaging, Stable Transfection, shRNA, Staining, Two Tailed Test, Western Blot, Expressing, Mutagenesis

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet: ( A ) DNA-RNA hybrids were quantified in undamaged U2OS cells by monitoring chromatin-bound RNaseH D10R-E48R -mCherry by FACS. 10,000 cells were counted in each of 3 biological replicates; error bars represent S.D. * and ** denote p < 0.05 or 0.01, respectively in Student's two-tailed T test with comparisons as indicated. ( B ) RNA/DNA hybrids were quantified in undamaged, CtIP-depleted U2OS cells complemented with either vector only, eGFP-CtIP(wt), or nuclease-deficient eGFP-CtIP(NA/HA) as in ( A ). 10,000 cells were counted in each of 3 biological replicates; error bars represent S. D. **** denotes p < 0.0001 using Student's two-tailed T test with comparisons as indicated. ( C ) S9.6 antibody was used to monitor RNA-DNA hybrids in wild-type or CtIP-depleted U2OS cells. Anti-Nucleolin was used as a marker for the nucleolus. ( D ) Quantification of S9.6 antibody signal in wild-type, CtIP-depleted, XPG-depleted, Senataxin-depleted, or double CtIP/XPG-depleted U2OS cells as indicated. Signal overlapping the nucleolin signal was excluded from the analysis; n > 50, Error bars represent S.E.M. *, **, and **** denote p < 0.05, 0.01, and 0.0001, respectively, using Student's two-tailed T test with comparisons as indicated. ( E ) Quantification of S9.6 antibody signal in wild-type, CtIP-depleted, or CtIP-depleted plus RNaseH overexpression in U2OS cells as in ( D ). ( F ) Wild-type or CtIP-depleted U2OS cells were exposed to CPT (5 µM) or were untreated before harvesting of cellular mRNA. Analysis of transcripts by RNA-seq and hierarchical clustering of transcripts from 21,412 genes is shown as a heat map (red for over-expressed, black for unchanged expression, and green for under-expressed genes) in comparison to wild-type undamaged cells (see ). ( G ) Statistical comparisons of overlap between the top 100 differentially expressed genes as ranked by DESeq differential expression p-value and DRIPc-seq peaks from GEO dataset GSE70189. Randomly picked genes were compared to this dataset (‘randomized control’) with the average of 35.56% and standard deviation 4.76% (estimated from 1000 simulations). Genes with significant differences between wild-type and CtIP-depleted cells were identified in the absence of DNA damage (‘WT vs CtIP shRNA (-CPT’)) as well as with CPT treatment (‘WT vs CtIP shRNA (+CPT’)) and were compared with the DRIPc-seq dataset. All three values are above the 99% confidence intervals for the null hypothesis that the genes showing the most evidence of differential expression overlapped the peak regions at the same rate as randomly selected genes.

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Two Tailed Test, Plasmid Preparation, Marker, Over Expression, RNA Sequencing, Expressing, Comparison, Quantitative Proteomics, Control, Standard Deviation, shRNA

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet: ( A ) RNA/DNA hybrids were quantified by DRIP-qPCR in U2OS cells containing a stably integrated, doxycycline-inducible transgene containing murine Sγ3 repeats. R-loop accumulation was measured by immunoprecipitation with the S9.6 antibody and qPCR for the Sγ3 region in the absence or presence of transcription (-/+Dox) in wild-type or CtIP-depleted cells; n > 6, error bars represent S.E.M. ( B ) Quantification of RNA/DNA hybrids in U2OS cells at endogenous loci using DRIP-qPCR. Levels of hybrids were measured at the ß-actin gene, with CtIP or XPG depletion, and with RNaseH overexpression in cells, as indicated; n > 3, error bars represent S. D. ( C ), ( D ), ( E ). DRIP-qPCR of sites in the RPL13A gene are shown with CtIP or XPG depletion and RNaseH overexpression in cells as indicated. In ( F ), CtIP-depleted cells were complemented with wild-type eGFP-CtIP or nuclease-deficient NA/HA mutant. ( G ) Senataxin ChIP was performed in cells containing the doxycycline-inducible transgene containing murine Sγ3 repeats. Levels of SETX occupancy were quantified in the presence or absence of doxycycline and with CtIP depletion as indicated and are represented as fold changes relative to wild-type cells in the absence of dox. n = 3, error bars represent standard deviation. * and ** denote p < 0.05 or 0.01, respectively, using Student's two-tailed T test with comparisons as indicated.

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Stable Transfection, Immunoprecipitation, Over Expression, Mutagenesis, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet: ( A ) Quantification of mRNA expression by RT-qPCR. A dox-inducible construct with murine Sγ3 repeats was stably integrated in U2OS cells, and the levels of the Sγ3 mRNA transcripts were measured in the absence or presence of transcription (dox) under wild-type or CtIP-depleted conditions. n > 6, Error bars represent S.D.

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Expressing, Quantitative RT-PCR, Construct, Stable Transfection

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet: ( A ) ( B ) Quantification of mRNA expression by RT-qPCR in U2OS cells with or without CtIP shRNA as indicated, measuring transcripts at the UBF and CD30 genes. ( C ) Quantification of mRNA expression in U2OS cells with or without CtIP shRNA as indicated, from the RNAseq dataset . ( D ) Quantification of mRNA expression in U2OS cells with or without CtIP shRNA as indicated, using RT-qPCR. Data shown is an average of 6 replicates, with error bars indicating S. D. * and *** denote p < 0.05 or 0.001, respectively, using Student's two-tailed T test with comparisons as indicated.

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Expressing, Quantitative RT-PCR, shRNA, Two Tailed Test

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet: ( A ) DNA breaks were quantified in wild-type and CtIP-depleted U2OS cells by alkaline comet assay. Cells were untreated (DMSO) or exposed to 5 µM CPT for 1 hr, with DRB (20 µM) or aphidicolin (2 µg/mL) pretreatment as indicated. Olive moments were calculated by analyzing at least 100 comets for each sample; error bars represent S.E.M. ( B ) Quantification of ssDNA breaks by alkaline comet assay was performed in wild-type, CtIP-depleted, XPG-depleted, or CtIP/XPG-depleted U2OS cells as in ( A ). ( C ) Quantification of ssDNA breaks by alkaline comet assay was performed in wild-type or CtIP-depleted U2OS cells complemented with wild-type eGFP-CtIP or nuclease-deficient NA/HA mutant CtIP as in ( A ). ( D ) A schematic representation of Ligation-mediated (LM)-PCR assay (top). Primer extension with a biotinylated primer (in red) from genomic DNA produces a double-stranded DNA end that is isolated with streptavidin and amplified by ligation-mediated PCR (asymmetric duplex and nested primers are presented in blue and green, respectively). LM-PCR assay measuring DNA breaks on the bottom strand of the RPL13A gene (bottom). DNA single-strand breaks were measured by LM-PCR at the endogenous RPL13A locus in wild-type or CtIP-depleted cells; n = 6, error bars represent S.E.M. * and **** denote p < 0.05 or 0.0001, respectively, using Student's two-tailed T test with comparisons as indicated.

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Alkaline Single Cell Gel Electrophoresis, Mutagenesis, Ligation, Isolation, Amplification, Two Tailed Test

Journal: eLife

Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

doi: 10.7554/eLife.42733

Figure Lengend Snippet:

Article Snippet: Cell line (human) , U2OS , ATCC , , .

Techniques: Transfection, Construct, Plasmid Preparation, shRNA, Sequencing