Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: Representative immunohistochemical staining of RAP80 positive expression in breast cancer tissue and paired normal breast tissue. Note: Positive nucleus staining of RAP80 in the paired normal breast mammary epithelium ( A , B ), RAP80 expression in ductal carcinoma in situ ( C ), and invasive ductal cancer ( D ); original magnification, all ×200.

Article Snippet: After blocking, blots were incubated with mouse anti-RAP80 monoclonal antibody (no 14466, diluted 1:400, Cellsignal) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology, Beijing, People’s Republic of China) overnight at 4°C, followed by each corresponding second antibody at room temperature for 1 hour at 37°C.

Techniques: Immunohistochemical staining, Staining, Expressing, In Situ

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: Expression of RAP80 in 162 patients with breast cancer by immunohistochemistry

Article Snippet: After blocking, blots were incubated with mouse anti-RAP80 monoclonal antibody (no 14466, diluted 1:400, Cellsignal) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology, Beijing, People’s Republic of China) overnight at 4°C, followed by each corresponding second antibody at room temperature for 1 hour at 37°C.

Techniques: Expressing, Amplification

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: Expression of RAP80 in 67 patients with breast cancer by qRT-PCR

Article Snippet: After blocking, blots were incubated with mouse anti-RAP80 monoclonal antibody (no 14466, diluted 1:400, Cellsignal) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology, Beijing, People’s Republic of China) overnight at 4°C, followed by each corresponding second antibody at room temperature for 1 hour at 37°C.

Techniques: Expressing, Amplification, Fluorescence In Situ Hybridization

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: Expression patterns of RAP80 in breast cancer cell lines. Notes: Total RNA and proteins were extracted from wild-type MCF-7, ZR-75, and MDA-MB-231, then subjected to qRT-PCR and Western blotting for RAP80. Both protein ( A – D ) and mRNA ( C , E ) expression of RAP80 were observed consistently in MCF-7, but ZR-75 and MDA-MB-231 had very weak RAP80 mRNA and protein expression. Therefore, we picked MCF-7 for transfection with RAP80 siRNA. Data are presented as mean ± SD of three independent experiments, * P <0.05 vs MCF-7 group. Abbreviations: NC, normal control; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: After blocking, blots were incubated with mouse anti-RAP80 monoclonal antibody (no 14466, diluted 1:400, Cellsignal) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology, Beijing, People’s Republic of China) overnight at 4°C, followed by each corresponding second antibody at room temperature for 1 hour at 37°C.

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

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: The Effect of RAP80 siRNA transfection on cell proliferation by MTT in wild-type MCF-7 or MCF-7 RAP80 siRNA. Notes: The survival rate of both cells decreased in a dose-dependent manner and the IC50 value for cisplatin in MCF-7 RAP80 siRNA cells was 0.83 µg/mL, and 1.69 µg/mL in wild-type MCF-7. Cell viability was measured by MTT; n=3 for each data point.

Article Snippet: After blocking, blots were incubated with mouse anti-RAP80 monoclonal antibody (no 14466, diluted 1:400, Cellsignal) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology, Beijing, People’s Republic of China) overnight at 4°C, followed by each corresponding second antibody at room temperature for 1 hour at 37°C.

Techniques: Transfection

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: RAP80 siRNA transfection decreased the invasive or migrating ability of MCF-7 breast cancer cells. Notes: Transwell migration assay shows the migrating ability of wild-type MCF-7 ( A ), MCF-7-siNC ( B ), and MCF-7 siRAP80 ( C ) at 72 hours after transfection. ( G ) Number of cells migrating onto the lower surfaces of the filter was counted. The transwell migration assay shows the invasive ability of wild-type MCF-7 ( D ), MCF-7-siNC ( E ), and MCF-7 siRAP80 ( F ) at 72 hours after transfection ( H ). Number of cells invading onto the lower surfaces of the filter was counted. Data are presented as mean ± SD of three independent experiments, * P <0.05 vs MCF-7 siRAP80 group. Abbreviation: NC, normal control.

Article Snippet: After blocking, blots were incubated with mouse anti-RAP80 monoclonal antibody (no 14466, diluted 1:400, Cellsignal) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology, Beijing, People’s Republic of China) overnight at 4°C, followed by each corresponding second antibody at room temperature for 1 hour at 37°C.

Techniques: Transfection, Transwell Migration Assay, Control

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: RAP80 siRNA transfection upregulated the expression of apoptotic proteins in MCF-7 cells. Notes: Western blot results showed that the siRNA transfection upregulated the protein expression of Caspase-3, cleaved Caspase-3, Apaf-1, Cytochrome C, Bax, and Fas, and it downregulated the mRNA and protein expression of Bcl-2. Protein levels analyzed by Western blot also confirmed this result ( A – G ). Data are presented as mean ± SD of three independent experiments, * P <0.05 vs MCF-7 siRAP80 group.

Article Snippet: After blocking, blots were incubated with mouse anti-RAP80 monoclonal antibody (no 14466, diluted 1:400, Cellsignal) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology, Beijing, People’s Republic of China) overnight at 4°C, followed by each corresponding second antibody at room temperature for 1 hour at 37°C.

Techniques: Transfection, Expressing, Western Blot

Journal: Molecular cell

Article Title: Fine-tuning of DNA damage-dependent ubiquitination by OTUB2 supports the DNA repair pathway choice.

doi: 10.1016/j.molcel.2014.01.030

Figure Lengend Snippet: Figure 1. Analysis of siRNA Screen Data (A) siRNAs from the siRNA library for human deubiquitinating enzymes were individually transfected into U2OS cells. These cells were treated with 5 ng/mL NCS and processed for RAP80 immunofluorescence staining 5 min after NCS treatment. Immunofluorescent images were obtained using an automated microscope, and the intensities of RAP80 foci were analyzed. Representative images from the screen are shown. Objective, 403. (B) Analyzed data from the screen. y axis: Total fluorescence intensity of RAP80 foci in each nucleus. x axis: Fluorescence intensity of each RAP80 focus. Three different nontargeting siRNAs were used as controls (CTRL). (C and D) Quantification of cells with 53BP1 foci. HeLa cells transfected with the indicated Flag-OTUB2 or Flag-OTUB1 expression plasmids were processed for 53BP1 and Flag immunofluorescence staining 1 hr after 5 ng/mL NCS treatment. The percentage of cells containing >10 53BP1 foci are shown. A total of 100 cells from the Flag-OTUB2- or OTUB1-transfected cultures with mild (C) or strong (D) fusion protein expression or 100 cells from the control cultures transfected with the Flag-empty plasmid were analyzed from each sample. Data are presented as the mean ± SD of three independent experiments. emp., empty plasmid. (E) Representative images of the immunofluorescence staining in (D) are shown. DNA was counterstained with DAPI. Scale bar, 25 mm. See also Figure S1.

Article Snippet: Antibodies We used the following antibodies: Flag (F3165, Sigma-Aldrich), DDDDK-tag (PM020, MBL), Myc (sc-40, Santa Cruz Biotechnology), conjugated ubiquitin (302-06751 [FK2], Nippon Biotest Laboratories), 53BP1 (NB100-305, Novus Biologicals; 612523 [19], BD Biosciences), RAP80 (NBP1-87156, Novus), RPA32 (NA18, Calbiochem) and RAD51 (70-001 lot 1, Bio Academia).

Techniques: Transfection, Staining, Microscopy, Fluorescence, Expressing, Control, Plasmid Preparation

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: Representative immunohistochemical staining of RAP80 positive expression in breast cancer tissue and paired normal breast tissue. Note: Positive nucleus staining of RAP80 in the paired normal breast mammary epithelium ( A , B ), RAP80 expression in ductal carcinoma in situ ( C ), and invasive ductal cancer ( D ); original magnification, all ×200.

Article Snippet: Control RAP80 siRNA (sc-92007, 1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and negative control siRNA (sc-36869, 1:500, Santa Cruz Biotechnology Inc.) were used in Western blot analysis to verify the downregulation of RAP80.

Techniques: Immunohistochemical staining, Staining, Expressing, In Situ

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: Expression of RAP80 in 162 patients with breast cancer by immunohistochemistry

Article Snippet: Control RAP80 siRNA (sc-92007, 1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and negative control siRNA (sc-36869, 1:500, Santa Cruz Biotechnology Inc.) were used in Western blot analysis to verify the downregulation of RAP80.

Techniques: Expressing, Amplification

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: Expression of RAP80 in 67 patients with breast cancer by qRT-PCR

Article Snippet: Control RAP80 siRNA (sc-92007, 1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and negative control siRNA (sc-36869, 1:500, Santa Cruz Biotechnology Inc.) were used in Western blot analysis to verify the downregulation of RAP80.

Techniques: Expressing, Amplification, Fluorescence In Situ Hybridization

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: Expression patterns of RAP80 in breast cancer cell lines. Notes: Total RNA and proteins were extracted from wild-type MCF-7, ZR-75, and MDA-MB-231, then subjected to qRT-PCR and Western blotting for RAP80. Both protein ( A – D ) and mRNA ( C , E ) expression of RAP80 were observed consistently in MCF-7, but ZR-75 and MDA-MB-231 had very weak RAP80 mRNA and protein expression. Therefore, we picked MCF-7 for transfection with RAP80 siRNA. Data are presented as mean ± SD of three independent experiments, * P <0.05 vs MCF-7 group. Abbreviations: NC, normal control; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: Control RAP80 siRNA (sc-92007, 1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and negative control siRNA (sc-36869, 1:500, Santa Cruz Biotechnology Inc.) were used in Western blot analysis to verify the downregulation of RAP80.

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

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: The Effect of RAP80 siRNA transfection on cell proliferation by MTT in wild-type MCF-7 or MCF-7 RAP80 siRNA. Notes: The survival rate of both cells decreased in a dose-dependent manner and the IC50 value for cisplatin in MCF-7 RAP80 siRNA cells was 0.83 µg/mL, and 1.69 µg/mL in wild-type MCF-7. Cell viability was measured by MTT; n=3 for each data point.

Article Snippet: Control RAP80 siRNA (sc-92007, 1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and negative control siRNA (sc-36869, 1:500, Santa Cruz Biotechnology Inc.) were used in Western blot analysis to verify the downregulation of RAP80.

Techniques: Transfection

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: RAP80 siRNA transfection decreased the invasive or migrating ability of MCF-7 breast cancer cells. Notes: Transwell migration assay shows the migrating ability of wild-type MCF-7 ( A ), MCF-7-siNC ( B ), and MCF-7 siRAP80 ( C ) at 72 hours after transfection. ( G ) Number of cells migrating onto the lower surfaces of the filter was counted. The transwell migration assay shows the invasive ability of wild-type MCF-7 ( D ), MCF-7-siNC ( E ), and MCF-7 siRAP80 ( F ) at 72 hours after transfection ( H ). Number of cells invading onto the lower surfaces of the filter was counted. Data are presented as mean ± SD of three independent experiments, * P <0.05 vs MCF-7 siRAP80 group. Abbreviation: NC, normal control.

Article Snippet: Control RAP80 siRNA (sc-92007, 1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and negative control siRNA (sc-36869, 1:500, Santa Cruz Biotechnology Inc.) were used in Western blot analysis to verify the downregulation of RAP80.

Techniques: Transfection, Transwell Migration Assay, Control

Journal: OncoTargets and therapy

Article Title: RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

doi: 10.2147/OTT.S186981

Figure Lengend Snippet: RAP80 siRNA transfection upregulated the expression of apoptotic proteins in MCF-7 cells. Notes: Western blot results showed that the siRNA transfection upregulated the protein expression of Caspase-3, cleaved Caspase-3, Apaf-1, Cytochrome C, Bax, and Fas, and it downregulated the mRNA and protein expression of Bcl-2. Protein levels analyzed by Western blot also confirmed this result ( A – G ). Data are presented as mean ± SD of three independent experiments, * P <0.05 vs MCF-7 siRAP80 group.

Article Snippet: Control RAP80 siRNA (sc-92007, 1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and negative control siRNA (sc-36869, 1:500, Santa Cruz Biotechnology Inc.) were used in Western blot analysis to verify the downregulation of RAP80.

Techniques: Transfection, Expressing, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: PKMYT1 kinase ameliorates cisplatin sensitivity in osteosarcoma

doi: 10.1038/s41392-025-02250-7

Figure Lengend Snippet: PKMYT1-induced NPM1 S260 phosphorylation promotes efficient DSB repair. a Western blot analysis of the expression of PKMYT1, phosphorylated-NPM1 S260, and phosphorylated-NPM1 T199 in OS cells after being treated with various concentrations of DDP. b – d Western blot analysis of the effect of DDP and IR on NPM1 S260 phosphorylation after PKMYT1 knockdown or inhibition with RP6306. e Western blot analysis of the impact of DDP and IR on NPM1 S260 phosphorylation following the overexpression of NPM1 S260 dephosphorylation mutant plasmid. f After transfection as indicated siRNAs for 48 h, HEK293T cells were collected and subjected to co-immunoprecipitation experiments followed by western blot analysis. g HEK293T cells were transfected with indicated siRNAs for 48 h and treated with or without cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. h HEK293T cells were treated with or without RP6306 (2 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. i HEK293T cells were treated with or without RP6306 (2 μM) and cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. j HEK293T cells were transfected with indicated plasmids for 24 h and treated with or without cisplatin (1 μM) before harvesting the cells for co-immunoprecipitation and Western blot. k – l Immunofluorescence analysis of the effect of NPM1 S260 phosphorylation on IR-induced BRCA1 foci. NPM1 knockout U2OS cells were rescued with either wild-type NPM1, NPM1 S260A mutant and NPM1 S260D mutant, with or without IR (10 Gy) treatment. Representative images are shown in k , and the number of BRCA1 foci per group was calculated in l. Approximately 100 cells were counted per group. Data are expressed as the mean ± SEM from three biological replicates. Statistical analysis was performed using Student’s t test, p value as indicated. Scale bar, 10 μm. m – n Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in m, and the number of BRCA1 foci per group was calculated in n, p value as indicated. Scale bar, 10 μm. o – p NPM1 knockout U2OS cells were rescued with indicated plasmids for 24 h, and immunofluorescence analysis of cells exposed to 10 Gy IR and recovered for 1 h. Representative immunofluorescence images were shown in o , and the number of RAD51 foci per group was shown in p , p value as indicated. Scale bar, 10 μm. q – r Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( q ), and the number of RAD51 foci per group was calculated in r . Scale bar, 10 μm. s , t Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( s ), and the number of RAP80 foci per group was calculated in t , p value as indicated. Scale bar, 10 μm. u HR assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with BRCA1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts. P value as indicated. v NHEJ assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with 53BP1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts, p value as indicated

Article Snippet: The antibodies used in this study are as follows: GAPDH (#60004-1-Ig, Proteintech, 1:10,000 dilution), PKMYT1 (#4282S, Cell Signaling Technology, 1:1,000 dilution), NPM1 (#60096-1-Ig, Proteintech, 1:5,000 dilution), p-NPM1 T199 (#AF3111, Affnity, 1:1000 dilution), p-NPM1 S260 (#TP50488, HUABIO, 1:3000 dilution), p-Histone H2A.X S139 (# P40705 , ProMab Biotechnologies Inc., China, 1:1000 dilution), RAP80 (#13642-1-AP, Proteintech, 1:1000 dilution), BRCA1 (#22362-1-AP, Proteintech, 1:1000 dilution), RAD51(#14961-1-AP, Proteintech, China, 1:2000 dilution), anti-HA (#AB0004, Abways, China, 1:2000 dilution), β-actin (A00730, Genscript Biotech, 1:1000 dilution), Phosphoserine/threonine/tyrosine (#11995 R, Yajikit, 1:100 dilution), HRP conjugated goat-anti-mouse antibody (#SA00001-1, Proteintech, 1:10,000 dilution) and HRP conjugated goat-anti-rabbit antibody (#511203, zen-bioscience, 1:5000 dilution).

Techniques: Phospho-proteomics, Western Blot, Expressing, Knockdown, Inhibition, Over Expression, De-Phosphorylation Assay, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Immunofluorescence, Knock-Out, Control, NHEJ Assay