Journal: bioRxiv

Article Title: Cancer-associated Fibroblast Spatial Heterogeneity and EMILIN1 Expression in Cancer Stroma Modulate TGF-β Activity and CD8 + T-Cell Infiltration in Breast Cancer

doi: 10.1101/2023.09.12.557312

Figure Lengend Snippet: EMILIN-1 is a good prognostic marker in breast cancer. (a) Multiplexed immunofluorescence analysis displaying the localization of CD8 + T cells (red) and EMILIN-1 expression (blue) in CAFs in a representative breast cancer sample (N=5). Expression of TGFBI, a TGF-β signaling activity marker, was in green. ( b ) Multiplexed immunohistochemistry analysis showing examples of EMILIN-1 (red) and CD8 (brown) co-staining in breast cancer samples (N=75, all subtypes; for multivariate analysis see Table S2 ). ( c ) Low-and high-power views of the multiplexed immunofluorescence analysis displaying the localization of EMILIN-1, Ki-67 and CD8 in representative breast cancer samples (N=7). ( d-e ) Violin plots of CD8 + cell counts and Ki-67 positivity in areas of high versus low EMILIN-1 expression in breast cancer samples (N=75). ( f ) Survival analysis of patients with breast cancer (N=75) in function of EMILIN-1 expression level (high versus low) in the tumor. Maybe you should remind what you used as cut-off for high/low levels.

Article Snippet: In the first five samples, EMILIN-1 staining (anti-EMILIN-1 rabbit polyclonal antibody: x400, HPA002822, Sigma) was visualized using the Opal 480 Fluorophore; CD8 staining (anti-CD8 rabbit polyclonal antibody: x500, ab4055, Abcam) with the Opal 570 Fluorophore; and TGFBI staining (anti-TGFBI rabbit polyclonal antibody: x400, 10188-1-AP; Proteintech, Rosemont, IL, USA) with the Opal 520 Fluorophore ( ).

Techniques: Marker, Immunofluorescence, Expressing, Activity Assay, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Ring finger protein 166 potentiates RNA virus-induced interferon-β production via enhancing the ubiquitination of TRAF3 and TRAF6

doi: 10.1038/srep14770

Figure Lengend Snippet: ( A ) RNF166 potentiates VISA- but not TBK1-mediated activation of the IFN-β promoter. 293T cells (2 × 10 5 ) were transfected with IFN-β reporter (50 ng), pRL-TK Renilla luciferase plasmid (50 ng), and expression plasmids for VISA or TBK1 together with an empty vector and RNF166 construct (50, 100, and 200 ng). Luciferase assays were performed 24 h after transfection. ( B ) RNF166 rather than RNF114, RNF125, and RNF138 potentiates the VISA-mediated activation of the IFN-β promoter and production of IFN-β. 293T cells (2 × 10 5 ) were transfected with the indicated plasmids. 24 h after transfection, the supernatants collected for IFN-β bioassays (right); Luciferase assays (left) were performed as in ( A ). ( C ) VISA- rather than TBK1-mediated activation of the IFN-β promoter was attenuated in 293T-shRNF166-#4 and #5 cells compared with 293T-shGFP cells. Luciferase assays were performed as in ( A ). ( D ) Overexpressed RNF166 interacts with overexpressed VISA, TRAF3, and TRAF6. 293T cells (1 × 10 7 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were immunoprecipitated (IP) with anti-HA (αHA). 24 h after transfection the immunoprecipitates and whole-cell lysates (WCL) were analyzed by western blot (IB) with anti-HA and anti-Flag antibody. ( E ) RNF166 co-localized with TRAF6 and TRAF3 but not VISA in HeLa cells. HeLa cells were transfected indicated expression plasmids (1 μg each). Immunofluorescent staining was performed with anti-Flag (red), anti-HA (green), and DAPI (blue). ( F ) Effects of SeV infection on the interaction between overexpressed RNF166 and endogenous TRAF3 and TRAF6. 293T cells (5 × 10 7 ) were transfected with Flag-RNF166 plasmids (5 μg each). 18 h after transfection, cells were infected with SeV for the indicated times. The cell lysates were immunoprecipitated (IP) and analyzed by western blot (IB) with the indicated antibodies. ( G ) RNF166 did not potentiate the VISA-mediated activation of the IFN-β promoter in 293T-shTRAF3 and 293T-shTRAF6 cells. Luciferase assays were performed as in ( A ). Each graph represents the mean ± SD of three independent experiments done in triplicate. ***indicates P < 0.001; **indicates P < 0.01; ns (not significant) indicates P > 0.05.

Article Snippet: Mouse antibodies against Flag and HA epitopes (Sigma-Aldrich), rabbit IgG (Sigma-Aldrich), IRDye800-conjugated anti-mouse and anti-rabbit IgG (Rockland Immunochemicals), rabbit polyclonal antibody against TRAF3 (Proteintech), rabbit polyclonal antibody against TRAF6 (Santa Cruz Biotechnology), mouse monoclonal antibody against GADPH (Abmart), poly(I:C) (Pharmacia), mouse polyclonal antibody against VISA (Hong-Bing Shu, Wuhan University, China), NDV-enhanced eGFP (Cheng Wang, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), Human Influenza A virus (Xin Ye, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China), EMCV (Xin Guo, China Agriculture University, Beijing, China), and HT1080 stably-transfected with ISRE luciferase construct (2fTGH) (Zheng-Fan Jiang, Peking University, China) were from the indicated sources.

Techniques: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Construct, Immunoprecipitation, Western Blot, Staining, Infection

Journal: Scientific Reports

Article Title: Ring finger protein 166 potentiates RNA virus-induced interferon-β production via enhancing the ubiquitination of TRAF3 and TRAF6

doi: 10.1038/srep14770

Figure Lengend Snippet: ( A ) 293T cell pools with stable knockdown of TRAF3, TRAF6, and VISA were generated by shRNA. Knockdown efficiency was analyzed by western blot with anti-TRAF3, TRAF6, and VISA antibodies. ( B ) SeV-induced IFN-β production was reduced when endogenous TRAF3, TRAF6, or VISA was stably knocked down. Indicated cells (5 × 10 5 ) were infected with SeV and IFN-β levels were estimated by Q-PCR ( B , left) and bioassay ( B , right) at the indicated times. ( C ) IRF3 dimer and p-IRF3 were attenuated in 293T-shTRAF3, 293T-shTRAF6, and 293T-shVISA cells infected with SeV. Indicated cells (5 × 10 5 ) were infected with SeV for the indicated times and lysates were analyzed by western blot with anti-p-IRF3 and anti-GADPH or by native western blot with anti-IRF3. ( D ) TRAF3 and TRAF6 knockout HEK293T cell lines were generated by CRISPER/CAS9. Knockout cells were analyzed by western blot with anti-TRAF3, TRAF6, and VISA antibodies. ( E ) p-IRF3 was attenuated in TRAF3-knockout (KO) 293T, TRAF6-KO 293T, and VISA-KO 293T cells infected with SeV. Indicated knockout cells (5 × 10 5 ) were infected with SeV and lysates were analyzed by western blot with anti-p-IRF3 antibody. ( F ) SeV-induced IFN-β was reduced when endogenous TRAF3, TRAF6, and VISA were knocked out. Q-RT-PCR (left) and IFN-β bioassay (right) were performed as in ( B ). Each graph represents the mean ± SD of three independent experiments done in triplicate. ***indicates P < 0.001; **indicates P < 0.01; ns (not significant) indicates P > 0.05.

Article Snippet: Mouse antibodies against Flag and HA epitopes (Sigma-Aldrich), rabbit IgG (Sigma-Aldrich), IRDye800-conjugated anti-mouse and anti-rabbit IgG (Rockland Immunochemicals), rabbit polyclonal antibody against TRAF3 (Proteintech), rabbit polyclonal antibody against TRAF6 (Santa Cruz Biotechnology), mouse monoclonal antibody against GADPH (Abmart), poly(I:C) (Pharmacia), mouse polyclonal antibody against VISA (Hong-Bing Shu, Wuhan University, China), NDV-enhanced eGFP (Cheng Wang, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), Human Influenza A virus (Xin Ye, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China), EMCV (Xin Guo, China Agriculture University, Beijing, China), and HT1080 stably-transfected with ISRE luciferase construct (2fTGH) (Zheng-Fan Jiang, Peking University, China) were from the indicated sources.

Techniques: Knockdown, Generated, shRNA, Western Blot, Stable Transfection, Infection, Bioassay, Knock-Out, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Ring finger protein 166 potentiates RNA virus-induced interferon-β production via enhancing the ubiquitination of TRAF3 and TRAF6

doi: 10.1038/srep14770

Figure Lengend Snippet: ( A ) Schematic structures of RNF166 and the deletion mutants used in this work. ( B , C ) Effects of truncations of RNF166 on SeV-induced ( B ) and VISA-mediated ( C ) activation of the IFN-β promoter. Transfection and luciferase assays were performed as above. ( D ) Effects of RNF166 and the RNF166 RING deletion mutant on VISA-mediated antiviral activity. 293T cells (5 × 10 5 ) were transfected with the indicated plasmids (1 μg). At 20 h after transfection, cells were infected with NDV-eGFP at an MOI of 0.0001, and at 40 h after infection, viral replication was determined by fluorescence microscopy. The GFP-positive cells were counted and the percentage was calculated. ( E ) Effects of RNF166 and the RNF166 RING deletion mutant on replication of SeV. 293T cells (5 × 105) were transfected with the indicated plasmids (1 μg). At 12 h after transfection, cells were infected with SeV, and at 24 h after infection, mRNA of P protein was determined by Q-PCR. ( F ) RNF166 interacted with TRAF3 and TRAF6 via its zinc-finger domain. Transfection and immunoprecipitation (IP) were performed as in ( D ). Each graph represents the mean ± SD of three independent experiments done in triplicate. ***indicates P < 0.001; **indicates P < 0.01; ns (not significant) indicates P > 0.05.

Article Snippet: Mouse antibodies against Flag and HA epitopes (Sigma-Aldrich), rabbit IgG (Sigma-Aldrich), IRDye800-conjugated anti-mouse and anti-rabbit IgG (Rockland Immunochemicals), rabbit polyclonal antibody against TRAF3 (Proteintech), rabbit polyclonal antibody against TRAF6 (Santa Cruz Biotechnology), mouse monoclonal antibody against GADPH (Abmart), poly(I:C) (Pharmacia), mouse polyclonal antibody against VISA (Hong-Bing Shu, Wuhan University, China), NDV-enhanced eGFP (Cheng Wang, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), Human Influenza A virus (Xin Ye, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China), EMCV (Xin Guo, China Agriculture University, Beijing, China), and HT1080 stably-transfected with ISRE luciferase construct (2fTGH) (Zheng-Fan Jiang, Peking University, China) were from the indicated sources.

Techniques: Activation Assay, Transfection, Luciferase, Mutagenesis, Activity Assay, Infection, Fluorescence, Microscopy, Immunoprecipitation

Journal: Scientific Reports

Article Title: Ring finger protein 166 potentiates RNA virus-induced interferon-β production via enhancing the ubiquitination of TRAF3 and TRAF6

doi: 10.1038/srep14770

Figure Lengend Snippet: ( A ) Overexpression of RNF166 enhanced the ubiquitination of overexpressed TRAF3/6. 293T cells (1 × 10 7 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were immunoprecipitated (IP) with anti-Flag (αF). The immunoprecipitates and whole-cell lysates (WCL) were analyzed by western blot (IB) with anti-HA and anti-Flag antibodies. ( B , C ) Knockdown of RNF166 inhibited SeV-mediated WT- and K63- but not K48-linked ubiquitination of endogenous TRAF3 ( B ) and TRAF6 ( C ). Indicated cells (3 × 10 7 ) were transfected with WT ubiquitin, K63O ubiquitin, or K48O ubiquitin (5 μg each). At 18 h after transfection, cells were infected with SeV for the indicated times. The lysates were immunoprecipitated with anti-TRAF3 (αTRAF3) or anti-TRAF6 (αTRAF6) antibody. The immunoprecipitates and whole-cell lysates (WCL) were analyzed by western blot (IB) with the indicated antibody. ( D , E ) Knockdown of RNF114 ( D ) and RNF138 ( E ) did not inhibit the SeV-mediated ubiquitination of endogenous TRAF3 and TRAF6. Transfection and immunoprecipitation were performed as in ( B ).

Article Snippet: Mouse antibodies against Flag and HA epitopes (Sigma-Aldrich), rabbit IgG (Sigma-Aldrich), IRDye800-conjugated anti-mouse and anti-rabbit IgG (Rockland Immunochemicals), rabbit polyclonal antibody against TRAF3 (Proteintech), rabbit polyclonal antibody against TRAF6 (Santa Cruz Biotechnology), mouse monoclonal antibody against GADPH (Abmart), poly(I:C) (Pharmacia), mouse polyclonal antibody against VISA (Hong-Bing Shu, Wuhan University, China), NDV-enhanced eGFP (Cheng Wang, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), Human Influenza A virus (Xin Ye, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China), EMCV (Xin Guo, China Agriculture University, Beijing, China), and HT1080 stably-transfected with ISRE luciferase construct (2fTGH) (Zheng-Fan Jiang, Peking University, China) were from the indicated sources.

Techniques: Over Expression, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Knockdown, Infection

Journal: Wellcome Open Research

Article Title: A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

doi: 10.12688/wellcomeopenres.16482.1

Figure Lengend Snippet: Scatter plot showing the enrichment of proteins in immunoprecipitations conducted using the Bethyl Labs anti-NCAPH2 (B276) antibody versus rabbit IgG control (n = 4, from cultures grown and processed in parallel), as determined by mass spectrometry and label-free quantification. The x-axis depicts mean Log2 fold-enrichment. The y-axis shows the Log10 probability of enrichment from Bayes-moderated t-tests. Condensin II and SWI/SNF subunits are labelled in blue and red, respectively.

Article Snippet: Each immunoprecipitation was performed using cells from ten 10cm plates, using 100 μL of Protein A Dyna beads (Invitrogen) precoated with 10 μg of Rabbit anti-NcapH2 (Bethyl Labs, A302-276A, lot 1) or rabbit IG (Cell Signalling Technology, #2729) and washed with PBS +0.01% tween.

Techniques: Mass Spectrometry

Journal: Wellcome Open Research

Article Title: A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

doi: 10.12688/wellcomeopenres.16482.1

Figure Lengend Snippet: Enrichment of Condensin II and SWI/SNF subunits in immunoprecipitations using B276 on crosslinked nuclear lysate.

Article Snippet: Each immunoprecipitation was performed using cells from ten 10cm plates, using 100 μL of Protein A Dyna beads (Invitrogen) precoated with 10 μg of Rabbit anti-NcapH2 (Bethyl Labs, A302-276A, lot 1) or rabbit IG (Cell Signalling Technology, #2729) and washed with PBS +0.01% tween.

Techniques:

Journal: Wellcome Open Research

Article Title: A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

doi: 10.12688/wellcomeopenres.16482.1

Figure Lengend Snippet: Enrichment of Condensin II and SWI/SNF subunits in immunoprecipitations using B276 on non-crosslinked nuclear lysate.

Article Snippet: Each immunoprecipitation was performed using cells from ten 10cm plates, using 100 μL of Protein A Dyna beads (Invitrogen) precoated with 10 μg of Rabbit anti-NcapH2 (Bethyl Labs, A302-276A, lot 1) or rabbit IG (Cell Signalling Technology, #2729) and washed with PBS +0.01% tween.

Techniques:

Journal: Wellcome Open Research

Article Title: A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

doi: 10.12688/wellcomeopenres.16482.1

Figure Lengend Snippet: A. Immunoblots (IB) show enrichment of the condensin subunit Smc4 and the SWI/SNF subunit Smarca4 in immunoprecipitations (IP) conducted from thymic whole cell extract using the B276 antibody compared to IgG control. B. Immunoblots show enrichment of the SWI/SNF subunit Arid1a (~270 KDa), but not the condensin II subunit Ncaph2 (~80 KDa), in immunoprecipitations conducted using an anti-Smarca4 antibody compared to IgG control. C. Schematic illustration of anti-FLAG affinity purification mass spectrometry strategy using Ncaph2 FLAG/FLAG mouse ESCs and untagged control. D. Scatter plot showing protein enrichment in the immunoprecipitation experiments shown in panel C (n = 4, from cultures grown and processed in parallel). The x-axis depicts mean Log2 fold-enrichment in Ncaph2 FLAG/FLAG (Flag) versus untagged control cells (WT). The y-axis shows the Log10 probability of enrichment from Bayes-moderated t-tests. Condensin II and SWI/SNF subunits are labelled in blue and red, respectively. Uncropped blots for panels A and B are available via Figshare .

Article Snippet: Each immunoprecipitation was performed using cells from ten 10cm plates, using 100 μL of Protein A Dyna beads (Invitrogen) precoated with 10 μg of Rabbit anti-NcapH2 (Bethyl Labs, A302-276A, lot 1) or rabbit IG (Cell Signalling Technology, #2729) and washed with PBS +0.01% tween.

Techniques: Western Blot, Affinity Purification, Mass Spectrometry, Protein Enrichment, Immunoprecipitation

Journal: Wellcome Open Research

Article Title: A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

doi: 10.12688/wellcomeopenres.16482.1

Figure Lengend Snippet: A . Schematic illustration of the auxin inducible degron system used to rapidly deplete NCAPH2 protein from HCT-116 cells. B . Immunoblots (IB) conducted on whole cell extract immunoprecipitated (IP) using the indicated antibodies. IPs were conducted in the presence (+) versus absence (-) of auxin-mediated NCAPH2 depletion. Uncropped blots are available via Figshare .

Article Snippet: Each immunoprecipitation was performed using cells from ten 10cm plates, using 100 μL of Protein A Dyna beads (Invitrogen) precoated with 10 μg of Rabbit anti-NcapH2 (Bethyl Labs, A302-276A, lot 1) or rabbit IG (Cell Signalling Technology, #2729) and washed with PBS +0.01% tween.

Techniques: Western Blot, Immunoprecipitation

Journal: Wellcome Open Research

Article Title: A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

doi: 10.12688/wellcomeopenres.16482.1

Figure Lengend Snippet: Antibodies used for immunoprecipitation and western blotting.

Article Snippet: Each immunoprecipitation was performed using cells from ten 10cm plates, using 100 μL of Protein A Dyna beads (Invitrogen) precoated with 10 μg of Rabbit anti-NcapH2 (Bethyl Labs, A302-276A, lot 1) or rabbit IG (Cell Signalling Technology, #2729) and washed with PBS +0.01% tween.

Techniques: Immunoprecipitation, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Methylation-associated silencing of BASP1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

doi: 10.1038/s12276-018-0067-4

Figure Lengend Snippet: a Expression of BASP1 is down-regulated in A/E-positive cell lines (Kasumi-1 and SKNO-1) compared to the other A/E-negative AML cell lines (NB4, U937, THP-1, KG-1, MV-4-11, HEL, and HL-60) and three sets of normal bone marrow (NBM). b Expression of BASP1 in different subtypes of AML and hematopoietic stem cells (HSC) analyzed from the GEO dataset (GSE13159). BASP1 expression was down-regulated in t(8;21) AML in contrast to the other subtypes of AML and HSC (*** p < 0.001). c The protein level of BASP1 was detected by western blotting in AML cell lines. β-actin was used as a control. d mRNA expression of BASP1 was plotted against that of AML1-ETO in A/E-positive AML ( n = 60) (GSE13159). The plot of BASP1 versus AML1-ETO expression showed an inverse correlation ( r = -0.38, p = 0.0027). e Upper panel: mRNA expression of BASP1 was measured in two pairs of cell lines: SKNO-1 and SKNO-1-siA/E, and U937 and U937-A/E. GAPDH was used as a control (** p < 0.01, *** p < 0.001). Lower panel: The protein levels of BASP1 were detected by western blotting. β-Actin was used as the control

Article Snippet: Rabbit polyclonal anti-BASP1 was purchased from Abcam (Cambridge, MA, USA, ab103315).

Techniques: Expressing, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Methylation-associated silencing of BASP1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

doi: 10.1038/s12276-018-0067-4

Figure Lengend Snippet: a Schematic diagrams of the CpG island of BASP1 promoter sequences (−2000 to −1 bp). The horizontal line (F1–R1) indicates the location of the primer used in the BSP assays. b Genomic bisulfate sequencing was performed to detect the methylation status of the DNA sequences in the CpG island of the BASP1 promoter from the indicated leukemia cell lines and normal bone marrow. Black dots and white dots indicate methylated and unmethylated CpG cytosine, respectively. c DNA methylation of BASP1 in nine A/E-positive AML cases (#) and three normal bone marrow samples (N) was demonstrated by MSP. M methylated allele, U unmethylated allele. d Upper panel: mRNA expression of BASP1 was up-regulated after continuous treatment with 0.5 or 1.0 µmol/L decitabine (DAC) for 3 days compared to the vehicle in Kasumi-1 and SKNO-1. GAPDH was used as a control. Error bars show the SD from three different assays (* p < 0.05, ** p < 0.01). Lower panel: The protein levels of BASP1 were detected by western blotting. β-Actin was used as a control

Article Snippet: Rabbit polyclonal anti-BASP1 was purchased from Abcam (Cambridge, MA, USA, ab103315).

Techniques: Sequencing, Methylation, DNA Methylation Assay, Expressing, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Methylation-associated silencing of BASP1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

doi: 10.1038/s12276-018-0067-4

Figure Lengend Snippet: a The left diagram shows a luciferase reporter containing various lengths of the BASP1 promoter region with wild-type A/E-binding sites – or mutated sequence (mut). Luciferase reporter assays showed that A/E repressed the transcriptional activity of the BASP1 promoter. b ChIP was performed in the SKNO-1 and SKNO-1-siA/E cell lines using anti-AML1, anti-ETO, anti-DNMT1, anti-DNMT3a, and anti-DNMT3b antibodies or a non-specific antibody (IgG control). Quantitative RT-PCR was performed with primers specific for the BASP1 promoter region surrounding the predicted −546/−556 binding site and expression was quantified as 2 −ΔCT relative to input. ChIP-PCR showed that A/E and DNMT3a bound the BASP1 promoter (* p < 0.05, ** p < 0.01)

Article Snippet: Rabbit polyclonal anti-BASP1 was purchased from Abcam (Cambridge, MA, USA, ab103315).

Techniques: Luciferase, Binding Assay, Sequencing, Activity Assay, Quantitative RT-PCR, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Methylation-associated silencing of BASP1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

doi: 10.1038/s12276-018-0067-4

Figure Lengend Snippet: a The CCK-8 assay was performed on SKNO-1 cells transduced with the GFP-tagged BASP1 lentiviral or GFP control plasmids at the indicated time points. b A colony formation assay was performed on SKNO-1 cells transduced with the GFP-tagged BASP1 lentiviral or GFP control plasmids and grown for 7 days. c Cell cycle analyses in SKNO-1 cells by flow cytometry showed that BASP1 overexpression induced the G1 phase arrest and blocked S-phase entry. d The induction of apoptosis in BASP1 overexpressing cells was measured using Annexin V-7AAD staining via flow cytometry. Error bars indicates the SD from three different assays (* p < 0.05)

Article Snippet: Rabbit polyclonal anti-BASP1 was purchased from Abcam (Cambridge, MA, USA, ab103315).

Techniques: CCK-8 Assay, Transduction, Colony Assay, Flow Cytometry, Over Expression, Staining