Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: Mouse anti-human MAD2 , Santa Cruz Biotechnology , Cat#sc-65492; RRID: AB_831526.

Techniques: Knock-Out, Two Tailed Test, Staining, Marker

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A New Method for Chromosomes Preparation by ATP-Competitive Inhibitor SP600125 via Enhancement of Endomitosis in Fish

doi: 10.3389/fbioe.2020.606496

Figure Lengend Snippet: Effects of SP600125 on the expression levels of spindle assembly checkpoint genes. (A) qRT-PCR analysis for the differentially expressed BUB1, MAD2 , and CDC20 genes between the control group and the SP600125-treated cells. (B) Western blot analysis of expression levels of BUB1, MAD2, and CDC20 upon SP600125 treatment, where “1” represents the cells in the control group (without SP600125), “2” stands for the cells treated by SP600125 for 48 h, and “3” represents the cells first being treated by SP600125 for 48 h, then by SP600125-free culture for the next 12 h, and further by SP600125 treatment for another 48 h. Each bar represents the mean ± SD of three independent experiments (* P < 0.05).

Article Snippet: The membrane was blocked with 5% (w/v) non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C and then incubated with primary antibodies, including anti-p53 (Abcam, USA), anti-MDM2 (Bioworld, USA), anti-p21 (Bioworld, USA), anti-Mad2L1 (Bioworld, USA), anti-CDC20 (Bioworld, USA), anti-BUB1 (Proteintech, USA), anti-Flag-tag (Proteintech, USA), and anti-β-ACTIN (Abcam, USA), overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A New Method for Chromosomes Preparation by ATP-Competitive Inhibitor SP600125 via Enhancement of Endomitosis in Fish

doi: 10.3389/fbioe.2020.606496

Figure Lengend Snippet: Analysis of cell proliferation by flow cytometry after transfection with pAd-MAD2. Lane 1 represents the normal cultured group. Lane 2 represents the negative control group. Lanes from 3 to 6 represent the overexpressed MAD2 groups (OE-MAD2): the cells being transfected with pAd-mCMV-MAD2-GFP-3-Flag-pA for 72 h (Lane 3), the cells being treated with SP600125 for the next 48 h (Lane 4), the cells continued to be further cultured in SP600125-free medium for 12 h (Lane 5), and the cells being further cultured using SP600125 treatment for another 48 h (Lane 6).

Article Snippet: The membrane was blocked with 5% (w/v) non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C and then incubated with primary antibodies, including anti-p53 (Abcam, USA), anti-MDM2 (Bioworld, USA), anti-p21 (Bioworld, USA), anti-Mad2L1 (Bioworld, USA), anti-CDC20 (Bioworld, USA), anti-BUB1 (Proteintech, USA), anti-Flag-tag (Proteintech, USA), and anti-β-ACTIN (Abcam, USA), overnight.

Techniques: Flow Cytometry, Transfection, Cell Culture, Negative Control

Journal: Cell death & disease

Article Title: FOXM1 is critical for the fitness recovery of chromosomally unstable cells.

doi: 10.1038/s41419-023-05946-2

Figure Lengend Snippet: Fig. 3 FOXM1 knockdown impairs fitness of MAD2-overexpressing cells. A Representative western blot and quantification of FOXM1 and HA-Mad2 in Kras (K1, K2, K3) and Kras/Mad2 (KM1, KM2, KM3) breast tumor cells. **P = 0.0043; Unpaired t-test. (n = 9 K and 9 KM tumors were analyzed and measured). B Quantitative RT-PCR of mouse Foxm1 expression in mammary tumors from K and KM animals (n = 8 K and n = 11 KM tumors). *P = 0.0338; Unpaired t-test. C Representative pictures (left) and relative cell viability (right) of K and KM tumor cells after siFoxm1 for 6 days (n = 6 K and n = 7 KM tumors with siFoxm1). Cell viability was normalized to untreated cells. *P = 0.0313; Unpaired t-test. D DNA content analysis of K and KM breast tumor cells after siFoxm1 for 6 days represented as the percentage of cells in each cell cycle phase. (n = 4 K and n = 6 KM tumors were analyzed). **P = 0.001, *P = 0.0304; Two-way ANOVA. E Percentage of TUNEL positive cells in K and KM tumor cells 6 days after Foxm1 knockdown (KD). (n = 4 K and 4KM tumors were analyzed). ***P = 0.0006; Two-way ANOVA. F Western blots of FOXM1, HA- Mad2, Cleaved-caspase3 (C-Casp3) and gama-H2AX in K and KM tumor cells after RCM-1 treatment or siFoxm1 for 6 days, performed in two biological replicates. ACTIN was used as a loading control. For original and additional wbs see Supplemental Material.

Article Snippet: Selection was performed with puromycin (1 μg/ml) or FAC sorting, then infected with inducible Tet-ON lentiviruses carrying human MAD2 and FOXM1 cDNA (MAD2 from Addgene #136347; FOXM1b from Addgene #68811; FOXM1c from Addgene #68810) and selected with hygromycin (300 μg/ml) or puromycin (1 μg/ml).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Expressing, TUNEL Assay, Control

Journal: iScience

Article Title: Distinct effects of sacituzumab govitecan and berzosertib on DNA damage response in ovarian cancer

doi: 10.1016/j.isci.2024.111283

Figure Lengend Snippet:

Article Snippet: Rabbit anti- MAD2L2/REV7 , ProteinTech , Cat #12683-1-AP; RRID: AB_2139530.

Techniques: Recombinant, Staining, Western Blot, Stripping, Control, SYBR Green Assay, cDNA Synthesis, Gene Expression, Software, Microscopy, Imaging

Journal: PLoS ONE

Article Title: Role of Cyclin B1/Cdc2 Up-Regulation in the Development of Mitotic Prometaphase Arrest in Human Breast Cancer Cells Treated with Nocodazole

doi: 10.1371/journal.pone.0024312

Figure Lengend Snippet: A . Changes in cell viability (MTT assay) after treatment of cells with 250 nM nocodazole for different lengths of time. Each data point is the mean ± S.D. from four replicate measurements from one representative experiment. B . Time-dependent induction of G 2 /M cell cycle arrest following treatment with nocodazole. Cells were seeded at 5×10 4 cells/mL and then treated with 250 nM nocodazole for 3, 6, 14, 24, 48 and 72 h. Cells were harvested and analyzed using flow cytometry. C . Cells were treatment with 250 nM nocodazole for 12 or 24 h, stained with Hoechst-33342, and examined in a phase contrast (PC) microscope (upper panel) or a fluorescence microscopy (lower panel) (at ×200 magnification). As shown, many cells are arrested in mitosis (prometaphase) after 250 nM treatment. D . Cells were treated with 250 nM nocodazole for 3, 6, 14, 24, 48 and 72 h. The morphology of cells arrested in prometaphase (based on 200 or more nuclei in each sample) was scored by fluorescence microscopy. Each bar is the mean ± S.D. value from three separate experiments. * P <0.05, ** P <0.01 versus vehicle-treated control. E ( upper part ). Time-dependent changes in cyclin B1 and Cdc2 protein levels following nocodazole treatment. Cells were treated with nocodazole (250 nM) for the length of time as indicated, and whole cell lysates were prepared. An equal amount of protein lysates was electrophoretically separated on the 10% SDS-polyacrylamide gel, and transferred to nitrocellulose membrane. Western blots were detected using specific antibodies against cyclin B1, Cdc2 (CDK1), MAD2, and Cdc20 on an enhanced chemiluminescence (ECL) apparatus. Membrane was stripped for determining the levels of GAPDH as a loading control. E ( lower part ). The relative protein levels for cyclin B1, Cdc2, MAD2, and Cdc20 were calculated according to their densitometry readings, which were normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate determinations. * P <0.05, ** P <0.01 versus vehicle-treated control. F . Cells were treatment with 250 nM nocodazole for 12 h and analyzed using immunofluorescence staining for cyclin B1 and Cdc2. Representative photographs were taken under a fluorescence microscope (original magnification, ×200).

Article Snippet: The cyclin B1 siRNAs (si-cyclin B1, catalog no. sc-29284, Santa Cruz), Cdc2 siRNAs (si-Cdc2, catalog no. sc-29252, Santa Cruz), MAD2 siRNAs (si-MAD2, catalog no. sc-35837, Santa Cruz), and the siRNA negative control (si-Con, catalog no. sc-37007, Santa Cruz) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: MTT Assay, Flow Cytometry, Staining, Microscopy, Fluorescence, Control, Membrane, Western Blot, Immunofluorescence

Journal: PLoS ONE

Article Title: Role of Cyclin B1/Cdc2 Up-Regulation in the Development of Mitotic Prometaphase Arrest in Human Breast Cancer Cells Treated with Nocodazole

doi: 10.1371/journal.pone.0024312

Figure Lengend Snippet: A . Cells were transfected with cyclin B1 siRNA (si-cyclin B1) and the negative control siRNAs (si-Con), 24 h later, cells were exposed to 250 nM nocodazole for additional 12 h. Then the whole cell lysates were analyzed for the levels of cyclin B1 and Cdc2by Western immunoblotting. B . Relative protein levels of cyclin B1 and Cdc2 are calculated according to densitometry readings, which are then normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate determinations. * P <0.05, ** P <0.01 versus vehicle-treated control; # P <0.05, ## P <0.01 versus nocodazole treatment. C . Cells were transfected with si-cyclin B1 or siRNA negative control and then further treated with nocodazole (250 nM) for 12 h. Cells with cyclin B1 knockdown were analyzed using immunofluorescent staining for cyclin B1. Representative photographs were taken using a fluorescence microscope (original magnification, ×200) or a phase contrast (PC) microscope (×200). D . Quantitative data on prometaphase-arrested cells. Each bar is a mean ± S.D. value from three separate experiments. * P <0.05, ** P <0.01 versus the vehicle-treated control; # P <0.05 versus nocodazole treatment. E . The DNA content of cells was analyzed using flow cytometry as described in the Material and Methods section.

Article Snippet: The cyclin B1 siRNAs (si-cyclin B1, catalog no. sc-29284, Santa Cruz), Cdc2 siRNAs (si-Cdc2, catalog no. sc-29252, Santa Cruz), MAD2 siRNAs (si-MAD2, catalog no. sc-35837, Santa Cruz), and the siRNA negative control (si-Con, catalog no. sc-37007, Santa Cruz) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Negative Control, Western Blot, Control, Knockdown, Staining, Fluorescence, Microscopy, Flow Cytometry

Journal: PLoS ONE

Article Title: Role of Cyclin B1/Cdc2 Up-Regulation in the Development of Mitotic Prometaphase Arrest in Human Breast Cancer Cells Treated with Nocodazole

doi: 10.1371/journal.pone.0024312

Figure Lengend Snippet: A . Cells were transfected with siRNA Cdc2 (si-Cdc2) and the negative control siRNAs (si-Con), and 24 h later, cells were exposed to 250 nM nocodazole for additional 12 h. Then the whole cell lysates were analyzed for Cdc2 and cyclin B1 levels using Western immunoblotting. B . The relative protein levels of Cdc2 and cyclin B1 are calculated according to their densitometry readings, which are normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate measurements. * P <0.05 versus vehicle-treated control; # P <0.05 versus nocodazole treatment. C . Cells were transfected with si-cyclin B1 or siRNA negative control and then further treated with nocodazole (250 nM) for 12 h. Cells with Cdc2 knockdown were analyzed using immunofluorescent staining. Representative photographs were taken using a fluorescence microscopy (original magnification, ×200) or a phase contrast microscope (×200). D . Quantitative data on prometaphase-arrested cells. Each bar is a mean ± S.D. value from three separate experiments. ** P <0.01 versus the vehicle-treated control; ## P <0.01 versus nocodazole treatment.

Article Snippet: The cyclin B1 siRNAs (si-cyclin B1, catalog no. sc-29284, Santa Cruz), Cdc2 siRNAs (si-Cdc2, catalog no. sc-29252, Santa Cruz), MAD2 siRNAs (si-MAD2, catalog no. sc-35837, Santa Cruz), and the siRNA negative control (si-Con, catalog no. sc-37007, Santa Cruz) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Negative Control, Western Blot, Control, Knockdown, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Role of Cyclin B1/Cdc2 Up-Regulation in the Development of Mitotic Prometaphase Arrest in Human Breast Cancer Cells Treated with Nocodazole

doi: 10.1371/journal.pone.0024312

Figure Lengend Snippet: A . Cells were transfected with siRNA MAD2 (si-MAD2) and the negative control siRNAs (si-Con), and 24 h later, cells were exposed to 250 nM nocodazole for additional 12 h. Then the whole cell lysates were analyzed for Cdc2, cyclin B1, MAD2, and Cdc20 levels using Western immunoblotting. B . The relative protein levels of Cdc2, cyclin B1, MAD2, and Cdc20 are calculated according to their densitometry readings, which are normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate measurements. * P <0.05 versus vehicle-treated control; # P <0.05 versus nocodazole treatment. C . Cells were transfected with si-MAD2 or siRNA negative control and then further treated with nocodazole (250 nM) for 12 h. Cells with Cdc2 knockdown were analyzed using immunofluorescent staining. Representative photographs were taken using a fluorescence microscopy (original magnification, ×200). D . Quantitative data on prometaphase-arrested cells. Each bar is a mean ± S.D. value from three separate experiments. ** P <0.01 versus the vehicle-treated control; # P <0.05 versus nocodazole treatment. E . The DNA content of cells was analyzed using flow cytometry as described in the Material and Methods section.

Article Snippet: The cyclin B1 siRNAs (si-cyclin B1, catalog no. sc-29284, Santa Cruz), Cdc2 siRNAs (si-Cdc2, catalog no. sc-29252, Santa Cruz), MAD2 siRNAs (si-MAD2, catalog no. sc-35837, Santa Cruz), and the siRNA negative control (si-Con, catalog no. sc-37007, Santa Cruz) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Negative Control, Western Blot, Control, Knockdown, Staining, Fluorescence, Microscopy, Flow Cytometry

Journal: PLoS ONE

Article Title: Role of Cyclin B1/Cdc2 Up-Regulation in the Development of Mitotic Prometaphase Arrest in Human Breast Cancer Cells Treated with Nocodazole

doi: 10.1371/journal.pone.0024312

Figure Lengend Snippet: Treatment of cancer cells with nocodazole causes microtubule disruption and thereby prevents microtubules from attaching to kinetochores in prometaphase cells. The unattached kinetochores will be bound by MAD2, which prevents the progression from prometaphase to metaphase and anaphase. It is speculated that the kinetochore-bound MAD2 protein plays an important role in mediating the up-regulation of cyclin B1 and Cdc2 proteins in prometaphase-arrested cells. The rapid increase of these two cell cycle proteins in prometaphase cells and particularly their accumulation in the nuclei are expected to be largely responsible for the development of characteristic nuclear phenotypes. Following a prolonged prometaphase arrest, the nocodazole-treated cells are expected to undergo cell death via intrinsic apoptosis pathways.

Article Snippet: The cyclin B1 siRNAs (si-cyclin B1, catalog no. sc-29284, Santa Cruz), Cdc2 siRNAs (si-Cdc2, catalog no. sc-29252, Santa Cruz), MAD2 siRNAs (si-MAD2, catalog no. sc-35837, Santa Cruz), and the siRNA negative control (si-Con, catalog no. sc-37007, Santa Cruz) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Disruption

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Establishment of a prognostic hypoxic gene signature in osteosarcoma in the TARGET cohort. (A) The chromosomal locations of 197 hypoxic genes. (B) The relationships between the number of trees and error rates. (C) The rank of relative importance of the top five variables. (D) Univariate cox regression analysis of TES, SDC3, MXI1, CAVIN1, and EGFR in osteosarcoma. (E) Heat map visualizing the up- (red) and down-regulated (green) genes in high- and low-risk groups. (F) The distributions and proportions of survival status (alive: green; dead: red). (G) Kaplan-Meier curves of OS between high- and low-risk specimens. The differences were compared by log-rank tests.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Univariate cox regression analysis for prognosis-related hypoxic genes in the TARGET cohort.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: The relative importance of prognosis-related hypoxic genes.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Prognostic values of hypoxic genes from the gene signature in osteosarcoma. Kaplan-Meier curves for overall survival of osteosarcoma patients in the high and low (A) CAVIN1, (B) EGFR, (C) SCD3, (D) TES, and (E) MXI1 expression groups from the TARGET cohort. The survival differences were assessed via log-rank tests.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Verification of the expression of hypoxic genes from the gene signature. (A–E) RT-qPCR for detecting the expression of SCD3, EGFR, MXI1, CAVIN1, and TES in U2OS osteosarcoma cells and hFOB1.19 normal cells. (F, G) Western blotting for the expression of the above genes in U2OS osteosarcoma cells and hFOB1.19 normal cells. (H, I) Western blotting for the expression of the above genes in Saos-2 osteosarcoma cells and hFOB1.19 normal cells. (J–N) RT-qPCR for the expression of the above genes in tumors with Saos-2 osteosarcoma cells and hFOB1.19 normal cells. ** p < 0.01; *** p < 0.001; **** p < 0.0001. U2OS osteosarcoma cells compared with hFOB1.19 normal cells.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: Development and Verification of a Hypoxic Gene Signature for Predicting Prognosis, Immune Microenvironment, and Chemosensitivity for Osteosarcoma

doi: 10.3389/fmolb.2021.705148

Figure Lengend Snippet: Silencing MXI1 attenuates proliferation, migration, and invasion of osteosarcoma cells. (A, B) RT-qPCR for verifying the expression of MXI1 in U2OS and Saos-2 cells following transfection with its specific siRNAs. (C, D) Colony formation for measuring the proliferative capacity of U2OS and Saos-2 cells after silencing MXI1 expression. (E–H) Transwell assays for detecting the (E, F) migration and (G, H) invasion of U2OS and Saos-2 cells with MXI1 knockdown. ** p < 0.01; **** p < 0.0001.

Article Snippet: Thereafter, the membranes were sealed by 5% BSA blocking buffer as well as incubated with primary antibodies against SCD (1:1,000; ab236868; Abcam, United States), EGFR (1:1,000; ab52894; Abcam), MXI1 (1:1,000; 12360-1-AP; Proteintech, Wuhan, China), CAVIN1 (1:1,000; ab76919; Abcam), TES (1:1,000; 10258-1-AP; Proteintech), and GAPDH (1:1,000; 6,004-1-lg; Proteintech) overnight at 4°C.

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, Knockdown

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Mad2 versus TRIP13 microarray expression data in three representative datasets from cBioPortal.org. (B) GGCGG CDE/E2F core sites present between -200 and +100 from the transcription start sites of MAD2, BUBR1, and TRIP13. (C) ChIP-Seq data from Encode database (https://genome.ucsc.edu/ENCODE/) for E2F1, E2F4, E2F6 binding near TRIP13 transcription start site in HeLa cells. (D) Mad2 and TRIP13 expression are both elevated by Western blot in Wild Type (WT) and Rb-family (Rb, p107, p130) deficient TKO MEFs. Quantification is shown below corresponding bands.

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Microarray, Expressing, ChIP-sequencing, Binding Assay, Western Blot

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Western blot of RPE2 cells transduced with dox-inducible HA-Mad2 construct and/or constitutive TRIP13-GFP construct with or without doxycycline addition for 24 hours. Total quantification (endogenous + exogenous) is shown below corresponding bands. (B) Mitotic timing of RPE-M or T13G cells, +/- 24 hours doxycycline, measured by cell rounding in time-lapse images. n ≥20 cells for each condition. (C) Equal numbers of RPE-M and T13G cells were plated with or without doxycycline on day 0 and were counted and replated on day 2 and day 4, and counted on day 8. Population doublings of RPE-M and T13G cells is plotted over time. n=3 independent experiments (D) Bright field imaging of RPE-M cells without doxycycline treatment and RPE-M and T13G cells with doxycycline treatment for 12 days. (E) Micronuclei were counted in 100 cells in triplicate with DAPI staining in RPE-M or T13G cells treated with doxycycline for 0, 1, 3, or 8 days. * indicates p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates not significant (p > 0.05); Unpaired t test. Data are represented as mean± SD.

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Western Blot, Transduction, Construct, Imaging, Staining

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Equal numbers of RPE-M cells transfected with TRIP13 or control siRNAs were plated with or without doxycycline on day 0 and were counted and replated on day 1 and counted on day 3. Population doublings of cells is plotted over time. n=3 independent experiments (B) Western blot of γH2AX and cleaved caspase 3 on RPE-M cells transfected with siRNAs for 6 days +/- Mad2 overexpression for 3 days. (C) Western blot of Mad2 and TRIP13 on parental RPE and Mad2-inducible RPE-M cells transduced with a doxycycline-inducible TRIP13 or control shRNA construct. Total quantification (endogenous + exogenous) is shown below corresponding bands. (D) Tumor volumes over time of RPE and RPE-M cells transduced with TRIP13 or control shRNA injected subcutaneously into nude mice (n=10). Mice were continuously fed with doxycycline-containing feed. Tumor volumes and incidence at week 5 are shown on the right. ** indicates p < 0.01; Unpaired t test. Data are represented as mean± SD.

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Control, Western Blot, Over Expression, Transduction, shRNA, Construct, Injection

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Validation of CRISPR-Cas9 knockout of TRIP13. Cell clones were screened for TRIP13 loss by Western blot. DNA from clone 2-19 was cloned and sequenced, with one allele having net deletion of seven base pairs and the second allele having a deletion of two base pairs, both leading to loss of reading frame. The gRNA sequence is indicated in bold. (B) Morphology of TRIP13 knockout cells by bright field microscopy (Left). Quantification of mitotic timing in RPE-M and TRIP13 knockout cells treated with +/- 200 ng/ml nocodazole 4 hours prior to imaging +/- 0.5 uM reversine 1 hour prior to imaging (Right). n ≥20 cells for each condition. (C) Morphology of TRIP13 knockout cells after 3 days Mad2 overexpression by bright field microscopy (Left). Quantification of mitotic timing and fate in RPE-M and TRIP13 knockout cells treated with +/- doxycycline 16 hours prior to imaging (Right). n ≥20 cells for each condition. (D) Mitotic timing of RPE-M or T13G cells treated with doxycycline for 24 hours or a low dose of nocodazole (15 ng/ml) 4 hours prior to imaging. n ≥20 cells for each condition. (E) Mitotic timing of RPE-M cells transfected with control siRNA or TRIP13 siRNA #1 4 days prior to imaging and treated with either doxycycline (24 hours) or low-dose (15 ng/ml) nocodazole (4 hours) prior to imaging. n ≥20 cells for each condition. ** indicates p < 0.01, *** p < 0.001, ns indicates not significant (p > 0.05); Unpaired t test. Data are represented as mean± SD. See also Figures S5 and S6

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Biomarker Discovery, CRISPR, Knock-Out, Clone Assay, Western Blot, Sequencing, Microscopy, Imaging, Over Expression, Transfection, Control

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Mitotic timing of TRIP13 wild-type, knockout (Left) or knockdown (Right) cells with or without Mad2-overexpression or reversine (0.5 uM) treatment. Mad2 induced 24 hours prior to imaging with doxycycline, reversine added 1 hour prior to imaging. n ≥20 cells for each condition. (B) Western blot of Cdc20 IPs from mitotic RPE-M and TRIP13 knockout cells +/- Mad2 overexpression +/- reversine. RPE-M and TRIP13 knockout cells were synchronized with 20 hours treatment of Cdk1 inhibitor RO-3306 +/- doxycycline 8 hours after 10 uM RO-3306 addition. Cells were released into fresh media for 30 minutes followed by treatment with 50 ng/ml nocodazole and 10 uM MG132 for 3 hours +/- 1 uM reversine for the last 1.5 hours. Mitotic cells were harvested by mitotic shakeoff. (C) Quantification of Mad2 in Cdc20 IPs in (B) normalized to Cdc20 for each condition (Left). Quantification of the reduction in normalized Mad2 levels in Cdc20 IP after reversine addition for each condition (Right). * indicates p < 0.05, ns indicates not significant (p > 0.05); Unpaired t test. Data are represented as mean± SD. See also Figure S7

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knock-Out, Knockdown, Over Expression, Imaging, Western Blot

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