Journal: Development (Cambridge, England)

Article Title: Genetic disruption of aurora B uncovers an essential role for aurora C during early mammalian development.

doi: 10.1242/dev.066381

Figure Lengend Snippet: Fig. 8. Genetic ablation of aurora B does not prevent mitotic arrest in the presence of nocodazole. (A)Duration of mitosis (in minutes) in primary Aurkb(lox/lox) or Aurkb(/) mouse embryo fibroblasts (MEFs) in the absence or in the presence of 3.5M nocodazole (Noc). Knock down of Mad2 expression by small interfering RNA (siRNA) was performed to inactivate the spindle assembly checkpoint (SAC) upon nocodozale treatment. (B)Mad2 protein levels are partially diminished in Aurkb(lox/lox) and Aurkb(/) cells, as shown by immunoblotting. (C)Localization of BubR1 and Mad2 to kinetochores in the presence of nocodazole. The ratio between BubR1 (n88 per genotype) or Mad2 (n22) and anti-centromere antibody (ACA; centromere) signals is shown. Representative images of BubR1 or Mad2 signals (green) in nocodazole-treated Aurkb(lox/lox) and Aurkb(/) cells. ACA is shown in red and DNA (DAPI) in blue. The - tubulin signal (red) indicates lack of microtubules in cells treated with 3.5M nocodazole. Scale bars: 10m.

Article Snippet: Embryos or MEFs were then blocked with 3% BSA and incubated with anti-centromere antibody (ACA; Antibodies Incorporated) or primary antibodies against -tubulin (Sigma), -tubulin (Sigma), aurora B (Abcam or BD Biosciences), BubR1 (a gift from S. S. Taylor, University of Manchester, UK), Mad2 [a gift of E. Salmon (University of North Carolina, USA) or K. Wassmann (CNRS, France)], cyclin B1 (Santa Cruz Biotechnology), incenp (a gift of C. Rodriguez, Hospital Puerta del Mar, Spain) and phospho-histone H3 Ser10 (P-H3; Upstate Biotechnologies).

Techniques: Knockdown, Expressing, Small Interfering RNA, Western Blot

Journal: Endocrinology

Article Title: Elucidating the Role of the Maternal Embryonic Leucine Zipper Kinase in Adrenocortical Carcinoma

doi: 10.1210/en.2018-00310

Figure Lengend Snippet: MELK expression associates with genes involved in cellular growth and division. (A) Increased MELK in H295R cells, compared with CU-ACC1 and CU-ACC2 cells, is associated with upregulation of BUB1B, PLK1, FOXM1 (**P < 0.01). (B) At the protein level, increased MELK correlated with increased expression of BUB1B, PLK1, FOXM1, and ERCC6L. (C and D) FOXM1 inhibition using thiostrepton was associated with a dose-dependent decrease in (C) FOXM1 and MELK mRNA and (D) FOXM1 and MELK protein expression. Densitometry analysis of the blots are shown in Supplemental Fig. 2B and 2C (**P < 0.01). 18S, 18S ribosomal RNA.

Article Snippet: Membranes were incubated with primary antibodies overnight, anti-MELK (Abcam, Cambridge, MA) ( 36 ), anti-PARP (Cell Signaling, Danvers, MA) ( 37 ), anti-Caspase-3 (Cell Signaling) ( 38 ), anti-Bub1b (Novus Biologicals, Littleton, CO) ( 39 ), and anti β -tubulin (Abcam) ( 40 ) were used at a dilution 1:1000; anti-FoxM-1 (Santa Cruz Biotechnology, Dallas, TX) ( 41 ), anti-ERCCL6 (Abcam) ( 42 ), and anti-PLK1 (Cell Signaling) ( 43 ) were used at a dilution of 1:500.

Techniques: Expressing, Inhibition

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Enhanced CDRs correlated with prostate cancer prognosis. (A) Visualization of core genes' dependency in different prostate cancer cells with CRISPR-Cas9 and siRNA screening. (B, C) Survival analysis of CDRs in different CRPC patient cohorts. (D) The relative expression of CDRs in normal prostate tissues and prostate cancer tissues in the TCGA-PRAD cohort. Gleason score and tumor stage correlation analysis of CDRs in different prostate cancer patient cohorts. All prostate patient cohorts were indicated in the corresponding panels. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: CRISPR, Expressing, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs linked with neuroendocrine features in prostate cancer cohorts. (A) The Venn diagram illustrates the genes specifically enhanced in prostate cancer compared with normal prostate tissues and NEPC compared with adenocarcinoma. (B, C) The relative expression of CDRs in NEPC compared with adenocarcinoma in different prostate cancer cohorts. (D) Correlation analysis of CDRs with NE score. The red indicates the higher NE score. (E, F) Pearson correlation analysis of CDRs with NE score CRPC cohort. (G) The heatmap indicates the relative expression of CDRs and AR, as well as KLK3, a transcription activity indication of AR. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NEPC, neuroendocrine prostate cancer; NE, neuroendocrine; AR, androgen receptor; KLK3, kallikrein-related peptidase 3.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, Activity Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs ablation suppressed prostate cancer cell proliferation. (A) The circuit diagram illustrates the correlation among CDRs co-expressed gene sets. (B) Venn analysis of the overlap of CDRs co-expressed genes. (C) The heatmap shows the relative expression of CDRs overlapped co-expressed genes in patients with NE signature high and low groups. (D – F) The heatmap illustrates the correlation of CDRs co-expressed genes with CDRs in ADPC (D) and CRPC (E, F) patient cohorts. (G, H) KEGG pathway analysis (G) and GSEA analysis (H) of enriched biological processes of CDRs and CDRs co-expressed genes. (I, J) The diagram illustrates the working model of CRISPR-Cas13 for RNA silencing and knockdown efficiency of CDRs with the corresponding gRNAs. (K, L) Cell growth assays indicate the impact of CDRs knockdown on the viability of different prostate cancer cells. (M) Western blotting analysis of the expression of cell cycle-regulated genes, including CCND1, CDK1, and p-CDK1, after CDRs knockdown. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NE, neuroendocrine; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; CCND1, cyclin D1; CDK1, cyclin-dependent kinase 1.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, CRISPR, Knockdown, Western Blot, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs were transcriptionally regulated by the RB1/E2F1 axis in prostate cancer. (A) The diagrams show the canonical E2F1 motif location within the promoter of CDRs. (B) ChIP-sequencing E2F1 enrichment peak within the promoter of CDRs. (C) The relative enrichment of E2F1 within the promoter of CDRs was determined using standard ChIP-qPCR. (D) ChIP-sequencing peaks show the relative enrichment of E2F1 in CDRs' promoters after RB1 is known. (E – G) The relative expression of CDRs in CRPC patients with different RB1 deletion status. (H) CDRs expression in patients with RB1 deletion mutations from different prostate cancer cohorts. (I) qRT-PCR detected the relative expression of CDRs after RB1 or E2F1 knockdown with CRISPR-Cas13. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). RB1, retinoblastoma tumor suppressor 1; E2F1, early 2 factor 1; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time PCR.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: ChIP-sequencing, ChIP-qPCR, Expressing, Quantitative RT-PCR, Knockdown, CRISPR, Ubiquitin Proteomics, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Virtual screening identified compounds that suppressed advanced prostate cancer. (A) The diagram illustrates structure-based virtual screening strategies for CDRs-targeted compounds. (B) The heatmap shows the binding affinity of compounds with CDRs. The red indicated a higher binding affinity of compounds with the corresponding compounds. (C, D) Cell viability assays were used to determine the tumor suppressive effect of compounds with high CDRs-binding affinity in different prostate cancer cell models. (E – G) The 2D structure of Q199, XDD60, and A79, which exhibits the most significant anti-tumor efficacy in prostate cancer cell models. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Compounds targeting CDRs exhibited superior anti-tumor efficacy compared with AR antagonists. (A – E) The tumor cell growth inhibition effects of different dosages of Q199, XDD60, and A79, as well as enzalutamide, were determined with CCK-8 assays (A–D), and the IC 50 of each agent was calculated with three independent experiments (E). (F, G) The histograms show the relative cell viability after being treated with 5 M of Q199, XDD60, or A79 alone, or a combination. (H) The Venn diagram shows the overlap of Q199, XDD60, and A79 potential targets predicted with SwissTargetPrediction ( http://swisstargetprediction.ch/ ). Molecular docking shows the binding of CDRs with Q199, XDD60, and A79. (I) The lowest binding (LB) affinity of CDRs with Q199, XDD60, and A79. ns, not significant. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). AR, androgen receptor.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Inhibition, CCK-8 Assay, Binding Assay, Ubiquitin Proteomics

Journal: Molecular Biology of the Cell

Article Title: Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

doi: 10.1091/mbc.E14-08-1330

Figure Lengend Snippet: Confinement interferes with the perpendicularity between the spindle pole axis and the metaphase plate during astral MT–mediated spindle alignment. (A) Sequential x-y-t / x-z-t confocal time-lapse series after the transition from prophase to metaphase in DRAQ5-stained MDCK cells expressing α-tubulin–GFP with flat and round shape. Note the increase of the maximum angle ε between the spindle axis (SA) and the metaphase plate axis (MA) in flat cells. (B) BUBR1, a spindle assembly checkpoint protein, was detected in prometaphase profiles and in metaphase profiles (white arrowheads) with high ε. (C) MDCK cells fixed and stained as indicated were grouped by their lower and upper z ′/ p ratios and C x-z (below and above quartiles Q1 and Q3, respectively) and their ε angle was determined. Thirty cells/experiment were analyzed for three independent experiments. (D) The ε angle was measured in cells from Supplemental Figure S7A. The NZ-washout population was grouped by its upper and lower z ′/ p ratios. (B, D) Yellow arrowheads indicate contacts between the cell apex and the metaphase plate. (C, D) Error bars indicate mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, analyzed by t test.

Article Snippet: The following antibodies were used in this study: α-tubulin (ab6160; Abcam, Cambridge, MA), γ-tubulin (T6557; Sigma, St. Louis, MO), BUBR1 (200-301-902; Rockland, Limerick, PA), and NuMA (ab36999; Abcam).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) Disease Free Survival or Overall Survival was evaluated in mCRPC patients with high BUB1B expression (top quartile) compared to patients with low BUB1B expression (bottom quartile) from (Cancer Cell, 2010) and SU2C RNA-seq data sets (downloaded from cBioportal) (log-rank test, ** p < 0.01, *** p < 0.001). (b) BUB1B mRNA expression was compared between normal or PC samples or between different disease stages in TCGA-PRAD (RNAseq data downloaded from http://xena.ucsc.edu/ ) MSKCC (Cancer Cell, 2010 from cBioportal), GSE94767, GSE32269, (microarray data from https://www.ncbi.nlm.nih.gov/gds ) (student’s t test, **** p < 0.0001). BUB1B protein was evaluated by IHC in (c) three pairs of LuCaP androgen dependent (AD) and castration resistant (CR) patient derived xenografts (PDXs) (LuCaP35, LuCaP77 and LuCaP96) or (d) in TMAs from metastatic CRPC samples from 10 patients with 16 bone and 17 visceral tissue (liver, spleen, lung and lymph node) (n=33) and benign tissue (n=2). Graphs represent the quantification of BUB1B positive cells IHC for (c) LuCaP PDXs and (d) TMAs. (student’s t test, * p < 0.05, ** p < 0.01). BUB1B (e) mRNA and (f) protein levels were evaluated in RWPE-1 [non-tumorigenic (NT), grey], LNCaP, VCaP (ADPC, yellow), C4-2B, 22Rv1, CWR-R1, PC3 and DU145 (CRPC, orange) cells. Graphs represent the average ± SEM of two or three independent experiments performed in triplicate.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Expressing, RNA Sequencing, Microarray, Derivative Assay

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) Cells excluding trypan blue were counted following transduction with shBUB1B (3’UTR) or control (shGFP) in LNCaP (AR positive) ADPC cells, C4-2B (AR positive), 22Rv1 (AR/ AR-V7 positive) and PC3 (AR-negative) CRPC cells. Graphs represent the average of four or five independent experiments performed in quadruplicate ± SEM. The area under the curve was calculated for each cell line (Mann-Whitney test, * p < 0.05, *** p < 0.001). (b) CDK1 and cyclin A levels after BUB1B depletion in 22Rv1 by Western blot. Western blot quantification is shown in Supplementary Figure 1 b. (c) Cell cycle distribution was measured after BUB1B depletion (vs control shGFP) in 22Rv1 cells by flow cytometry of propidium iodide (PI) stained cells. Graphs represent distribution of the cell population across the cell cycle of the average ± SEM of four independent experiments (Mann-Whitney test, * p < 0.05). (d) Representative images of mitosis duration evaluated by live cell imaging using the Incucyte Zoom System in 22Rv1 cells after BUB1B depletion (vs control shGFP). Graphs show quantification of one representative experiment (50 randomly chosen cells per group), of two independent experiments (unpaired t-test, * p < 0.05).

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Transduction, Control, MANN-WHITNEY, Western Blot, Flow Cytometry, Staining, Live Cell Imaging

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) BUB1B levels were determined by western blot of lysates from LNCaP and VCaP ADPC cells ectopically expressing BUB1B or EV. (b) LNCaP and VCaP BUB1B vs EV cell proliferation was measured by trypan blue exclusion in the absence of androgens after eight days. Graphs represent the average ± SEM of three or four independent experiments performed in triplicate (Mann-Whitney test, * p < 0.05). (c) Colony formation was evaluated in LNCaP EV and BUB1B in soft agar assays after fifteen days. Images are representative of one of three independent experiments. Graphs represent the average of three independent experiments performed in triplicate (unpaired t-test, **** p < 0.0001). (d) LNCaP EV or BUB1B expressing cells were subcutaneously xenografted in castrated SCID mice (n=10 for EV and n=29 for BUB1B) and tumor volumes (mm 3 ) were measured by caliper every 3 days for 85 days. Tumor volume over time was evaluated using a mixed model with cubic effect. Differences were observed between groups at the end of the experiment (day 85). (e) End point PSA in plasma was determined by ELISA (Mann-Whitney test, **** p < 0.0001) (f) Images of end-stage tumors from LNCaP EV and BUB1B xenografts (scale bar = 1 cm). (g) BUB1B was evaluated by western blot in tumor tissue extracts. Tumor images and the corresponding tumor samples were ordered based on tumor size. (h) Graph shows the correlation between tumor weight and BUB1B expression (Pearson correlation, ** p < 0.01).

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Western Blot, Expressing, MANN-WHITNEY, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) LNCaP and VCaP ADPC cells expressing BUB1B (WT), D882N, K795R (kinase dead mutants) or control cells (EV) were grown in the absence of androgen and BUB1B protein was evaluated by western blot. Growth of LNCaP and VCaP expressing EV, BUB1B (WT), D882N and K795R was determined by trypan blue after eight days in culture. Graphs represent the average of three independent experiments performed in triplicate. (Kruskal-Wallis test, * p < 0.05, ** p < 0.01). (b) Colony formation of LNCaP EV, BUB1B, D882N and K795R was evaluated by soft agar assays after 3 weeks. Images are representative of three independent experiments (one-way ANOVA, * p < 0.05, **** p < 0.0001). BUB1B, D882N,K795R or EV control cells were expressed in 22Rv1 CRPC. (c) 22Rv1 cell proliferation was measured by trypan blue exclusion assay after BUB1B knockdown (siRNA against non-coding sequences) and ectopic expression of BUB1B, D882N,K795R or EV control. The graph represents the average of four independent experiments performed in triplicate (unpaired t-test, **** p < 0.0001). The lower panel shows BUB1B protein by western blotting in 22Rv1 EV, BUB1B, D882N and K795R expressing cells after knockdown of endogenous BUB1B using siBUB1B RNA targeting the 3’UTR.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Expressing, Control, Western Blot, Trypan Blue Exclusion Assay, Knockdown

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) AR was evaluated by western blot in LNCaP EV and BUB1B cells growing in castrated conditions (2% CSS) at 0, 4 and 8 days. (b) AR was knocked down in LNCaP EV or BUB1B cells by siRNA and cell number was quantified by trypan blue exclusion assay after eight days. Graph represents three independent experiments performed in triplicate (Mann-Whitney test, * p < 0.05). AR levels after AR knockdown using siRNA in LNCaP EV and BUB1B cells are shown.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Western Blot, Trypan Blue Exclusion Assay, MANN-WHITNEY, Knockdown

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) Cell proliferation determined by trypan blue exclusion after eight days of enzalutamide treatment (10 µM) in LNCaP and VCaP EV and BUB1B cells. The graph represents three independent experiments done in triplicate (Mann-Whitney test, * p < 0.05). (b) Colony formation in soft agar was measured after fifteen days. Images are representative of three independent experiments performed in triplicate. Graphs are representative of three to four independent experiments performed in triplicate (one-way ANOVA, ** p < 0.01, **** p < 0.0001). (c) BUB1B was evaluated by western blot in LNCaP xenograft derived T_EV and T_BUB1B cells. (d) T_EV and T_BUB1B cells were grown in the absence of androgens and proliferation was measured by trypan blue exclusion assays. The graph represents three independent experiments performed in triplicates. The area under the curve was calculated (unpaired t-test, * p < 0.05). (e) Proliferation of T_EV and T_BUB1B cells treated with 10 µM enzalutamide (or vehicle) for eight days is shown. The graph represents three independent experiments performed in triplicate (Kruskal-Wallis test, * p < 0.05). (f) LNCaP EV or T_BUB1B expressing cells were subcutaneously xenografted into castrated SCID mice (EV n=10 and T_BUB1B n=29) and tumor volumes (mm3) were measured by caliper every 3 days. Once tumors reach 200 mm3, animals were split into two groups and treated daily with enzalutamide (n=9) or vehicle (n=11). Graph shows tumor volume over time. (g) PSA levels in plasma were determined by ELISA (Mann-Whitney test, **** p < 0.0001). (h) Images of end-stage tumors from T_BUB1B vehicle (n=11) or enzalutamide (n=9) xenografts are shown. (i) BUB1B protein was evaluated in tumors by western blot. Tumor images and protein samples were ordered based on tumor size, vehicle tumors #1 and 2 were repeated in both blots for comparison.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: MANN-WHITNEY, Western Blot, Derivative Assay, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: Cell proliferation was measured after GSK-923295 (CENP-E inhibitor) treatment (0, 0.03, 0.06, 0.12 and 0.25 µM) for 7 days in (a) LNCaP EV and BUB1B cells or (b) LNCaP T_EV and T_BUB1B cells. Graph represents four independent experiments performed in triplicate (one-way ANOVA, * p < 0.05, ** p < 0.01). (c) BUB1B cells growing in castration conditions were treated with GSK-923295 (10-30 nM), enzalutamide (10-30 µM) or the combination for 7 days and cell number was evaluated by trypan blue exclusion assay. Two independent experiments were performed in triplicate. Combinatory index (CI) was calculated using the CompuSyn software. A CI < 1 indicates synergism, = 1 indicates additivity,> 1 indicates antagonism.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Trypan Blue Exclusion Assay, Software

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) LNCaP and VCaP ADPC cells expressing BUB1B (WT), D882N, K795R (kinase dead mutants) or control cells (EV) were grown in the absence of androgen and BUB1B protein was evaluated by western blot. Growth of LNCaP and VCaP expressing EV, BUB1B (WT), D882N and K795R was determined by trypan blue after eight days in culture. Graphs represent the average of three independent experiments performed in triplicate. (Kruskal-Wallis test, * p < 0.05, ** p < 0.01). (b) Colony formation of LNCaP EV, BUB1B, D882N and K795R was evaluated by soft agar assays after 3 weeks. Images are representative of three independent experiments (one-way ANOVA, * p < 0.05, **** p < 0.0001). BUB1B, D882N,K795R or EV control cells were expressed in 22Rv1 CRPC. (c) 22Rv1 cell proliferation was measured by trypan blue exclusion assay after BUB1B knockdown (siRNA against non-coding sequences) and ectopic expression of BUB1B, D882N,K795R or EV control. The graph represents the average of four independent experiments performed in triplicate (unpaired t-test, **** p < 0.0001). The lower panel shows BUB1B protein by western blotting in 22Rv1 EV, BUB1B, D882N and K795R expressing cells after knockdown of endogenous BUB1B using siBUB1B RNA targeting the 3’UTR.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Expressing, Control, Western Blot, Trypan Blue Exclusion Assay, Knockdown

Journal: ACS sensors

Article Title: Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-μFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

doi: 10.1021/acssensors.3c01060

Figure Lengend Snippet: Figure 5. Uni-μFCM was combined with gene knockout to measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies. (A) Scatter plots of XRCC3, Cdc20, and BubR1 from paired tumor cell lines of salivary adenoid cystic carcinoma with low (SACC-83) and high (SACC-LM) lung metastatic capabilities. (B) Scatter plots of single- cell expressions of XRCC3, Cdc20, and BubR1 from colon tumor cell lines HCT116 P53+/+ and its subtype HCT116 P53−/−with P53 deficiency.

Article Snippet: Fluorescence-labeled antibodies used for cell staining included antibodies of OCT4-Alexa 488 purchased from Thermo Fisher, USA, CD3-Alexa 488, XRCC3-Alexa 488, c-Myc-PE, CD79a-PE, Cdc20-PE, KLF4-PerCP, LAMP1-PerCP, and BubR1-PerCP purchased from NOVUS, USA.

Techniques: Gene Knockout