Journal: Aging Cell

Article Title: Age‐dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B

doi: 10.1111/acel.13489

Figure Lengend Snippet: SAC mediator and scaffolding proteins are reduced at kinetochores in Aurkb cKO oocytes from older females. (a, c, e, g, I, k) Representative confocal images of oocytes from wild‐type (WT) or conditional Aurkb knockouts (B cKO) from young and older females. Centromeres were detected by staining with antibodies against ACA (red) and chromosomes were detected with DAPI‐staining (blue). (a, c) Representative confocal images of Met I oocytes immunostained with antibody against MAD2 (gray) from young (a) or older females (c). (b) Quantification of MAD2 intensity at kinetochores in (a) ( p = 0.2711; number of oocytes examined, WT: 51, B cKO: 36; 4 mice/genotype). n.s.: not significant. (d) Quantification of MAD2 intensity at kinetochores in (c) (One‐way ANOVA, **** p < 0.0001; number of oocytes examined, WT: 52, B cKO: 47, C KO: 39; 3 mice/genotype). (e) Early pro‐metaphase I oocytes immunostained with antibodies against MAD2 (gray). (f) Quantification of MAD2 intensity at kinetochores in (e) (**** p < 0.0001; number of oocytes examined, WT: 40, B cKO: 35; 6 mice/genotype). (g) Late pro‐metaphase I oocytes immunostained with anti‐MPS1 (gray); (h) Quantification of MPS1 intensity at kinetochores of (g) ( p = 0.0514; number of oocytes examined, WT: 31, B cKO: 33; 3 mice/genotype). (i, k) Representative confocal images of metaphase I oocytes immunostained with (i) BUB1 (gray) or (k) ZW10 (gray). (j) Quantification of BUB1 intensity at kinetochores showed in (i) (**** p < 0.0001; number of oocytes examined, WT: 39, B cKO: 37; 3 mice/genotype). (l) Quantification of ZW10 intensity at kinetochores showed in (k) (* p <0.0121; number of oocytes examined, WT: 40, B cKO: 27; 3 mice/genotype). n.s.: not significant. Examples of individual bivalent (boxes) are magnified and shown in the zoom panels. Scale bars: 10 μm and 2 μm. Graph shows the mean value per oocyte of at least 30 kinetochores measured for each oocyte and includes the mean ±SEM from 3 experiments. Except for panels c‐d, Unpaired Students t‐Test, two‐tailed used

Article Snippet: Human MAD2 was amplified by PCR and cloned into the pIVT–Gfp vector (Addgene #16047).

Techniques: Scaffolding, Staining, Two Tailed Test

Journal: Aging Cell

Article Title: Age‐dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B

doi: 10.1111/acel.13489

Figure Lengend Snippet: Aurkb cKO oocytes from older females have reduced expression of MAD2, ZW10 and Securin. (a) Representative Western blot images of prophase I‐arrested oocytes from older wild‐type (WT) or conditional Aurkb knockout (B cKO) females detecting MAD2, ZW10 and Securin. (b) Quantification of MAD2 (** p < 0.0030), (c) Quantification of ZW10 (** p < 0.0016). (d) Quantification of Securin (* p < 0.0468). (e, g) Representative Western blots of prophase I‐arrested oocytes from young females detecting MAD2, Securin (d) and ZW10 (g). (f) Quantification of MAD2 ( p = 0.9774). Graph shows individual values plus the mean ±SEM from 3 independent experiments. (h) Quantification of ZW10 ( p = 0.1166). (i) Quantification of Securin (* p < 0.7505), MAD2, ZW10 and Securin signals were normalized to α‐Tubulin. Each lane contained 100 prophase I‐arrested oocytes. n.s.: not significant. Graph shows mean ±SEM from 2–3 independent experiments. (j) Representative confocal images of pro‐metaphase I oocytes expressing Gfp or MAD2‐gfp (green) DNA (blue). Scale bar: 10 μm. (k) Representative images of timing of PBE of Aurkb cKO oocytes from older females, matured in nocodazole expressing either Gfp or MAD2‐gfp. Red star indicates a PB. Scale bar: 20 μm. (l) Quantification of % of oocytes from (k) that undergo PBE in nocodazole. (* p = 0.0.0160; number of oocytes examined Gfp: 73, MAD2‐gfp: 59; 6 mice). Graph shows the mean ±SEM from 3 experiments. (m) Representative images of timing of PBE of Aurkb cKO oocytes from older females, matured in nocodazole expressing Securin‐gfp (green). Numbers of oocytes that expelled a PB relative to total number of oocytes analyzed is indicated to the right of the images. Unpaired Students t‐Test, two‐tailed was used

Article Snippet: Human MAD2 was amplified by PCR and cloned into the pIVT–Gfp vector (Addgene #16047).

Techniques: Expressing, Western Blot, Knock-Out, Two Tailed Test

Journal: Aging Cell

Article Title: Age‐dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B

doi: 10.1111/acel.13489

Figure Lengend Snippet: ROS levels are increased in Aurkb cKO oocytes from older females. (a) Representative images of CM‐H2DCFDA fluorescence in prophase I‐arrested oocytes from wild‐type (WT), conditional Aurkb knockouts (B cKO), or Aurkc knockout (C KO) females at different ages. (b) Quantification of CM‐H2DCFDA intensity showed in (a) **** p < 0.0001; number of oocytes examined: positive control: 29; young WT: 58, B cKO: 77, C KO: 92; older: WT: 60, B cKO: 36, C KO: 70; 3 mice/genotype). (c) Representative images of CM‐H2DCFDA fluorescence in prophase I‐arrested oocytes from wild‐type (WT), conditional Aurkb knockouts (B cKO), older females treated with and without NAC. Positive control: WT oocytes incubated with 200 μM H 2 O 2 . Scale bars: 20 μm. (d) Quantification of CM‐H2DCFDA intensity showed in (c) (** p = 0.0035; * p < 0.05; number of oocytes examined: WT: 20, B cKO: 25, B cKO +NAC:29; 3 mice/genotype). (e) Representative confocal images of WT and Aurkb cKO Met I oocytes from older females treated with or without NAC, immunostained with antibodies against ACA (red), MAD2 (gray) and DAPI (blue). (f) Quantification of MAD2 intensity at kinetochores in (e) (* p = 0.0464; **** p < 0.0001; n.s. not significant; number of oocytes examined: WT: 35, B cKO: 32, B cKO +NAC:28; 3 mice/genotype). Graph shows individual oocyte values plus the mean ±SEM from 3 experiments. One‐way ANOVA used

Article Snippet: Human MAD2 was amplified by PCR and cloned into the pIVT–Gfp vector (Addgene #16047).

Techniques: Fluorescence, Knock-Out, Positive Control, Incubation

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: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Hec1 S69 is phosphorylated throughout mitosis. (A) Amino acid sequences of the human and PtK1 cell Hec1 N-terminal tail domain. Shown in yellow is the human peptide sequence that was used to generate the S69 phosphospecific antibody. The arrow points to S69 in the human sequence and the corresponding serine residue in the PtK1 sequence. Asterisks indicate all other mapped Aurora B kinase sites in the human Hec1 tail domain ( ; ). (B) Immunofluorescence images of HeLa cells stained with phosphospecific antibodies to Hec1 pS69. Depletion of Hec1 (bottom) results in loss of pS69 staining at kinetochores. Cells are also immunostained with antibody 9G3 (pan-Hec1 antibody) and an anticentromere antibody (ACA) derived from human calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum. (C) Immunofluorescence images of HeLa cells demonstrating kinetochore localization of pS69 during mitosis. Quantification is shown on the right. For each phase shown, ≥400 kinetochores from ≥30 cells were measured. (D) Immunofluorescence images of a HeLa cell stained with antibodies to pS69 and Mad2. For the cell shown, most chromosomes are aligned at the spindle equator, and one chromosome remains near a spindle pole (arrows). A schematic illustrating examples of pole-proximal chromosomes is shown on the left. (E) Immunofluorescence images of HeLa cells depleted of CENP-E to increase the number of pole-proximal chromosomes (arrows) and stained with Hec1 phosphospecific antibodies. Quantification is shown on the right from one representative experiment. n values are as follows: pS69, 20 polar kinetochores and 40 aligned kinetochores; pS55, 17 polar kinetochores and 57 aligned kinetochores; and pS44, 13 polar kinetochores and 29 aligned kinetochores. Error bars indicate SD. Bars: (B, C, and E) 10 µm; (D) 3 µm.

Article Snippet: Two rabbits were immunized with the Mad2 protein (Rockland Immunochemicals Inc), and antisera from the two injected rabbits were affinity purified against full-length Mad2 protein using a HiTrap-NHS column.

Techniques: Sequencing, Residue, Immunofluorescence, Staining, Derivative Assay

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: Acta biomaterialia

Article Title: Overcoming Cisplatin Resistance in Non-Small Cell Lung Cancer with Mad2 Silencing siRNA Delivered Systemically using EGFR-Targeted Chitosan Nanoparticles

doi: 10.1016/j.actbio.2016.09.045

Figure Lengend Snippet: (A) qPCR and (B) western blot analysis of Mad2 knockdown efficiency in A549 WT and A549 DDP tumors. A 3 mg/kg single dose of siRNA, encapsulated either in TG or NTG NPS, was administered to tumor bearing mice. Tumors were collected at different time points for evaluation of the Mad2 gene silencing. (n = 5 animals/group). On qPCR data, bars represent the mean ± standard deviation from three independent experiments. *** = p< 0.01. Referring to the western data, tubulin protein was used as an internal control. Below each set of western blot are the values of the relative density of Mad2 protein levels, which were normalized to those of tubulin. Data represents one of the three independent experiments with similar results. DDP: Cisplatin; NTG: Non Targeted; SCR, scramble sequence siRNA; SD: Standard Deviation; TG: Targeted; WT: Wild Type

Article Snippet: A pool of 3 sequences of siRNA duplexes targeted against Mad2 mRNA (CAT. sc-35837) and a non-targeting negative control duplexes (CAT. sc-37007) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX).

Techniques: Western Blot, Knockdown, Standard Deviation, Control, Sequencing

Journal: bioRxiv

Article Title: Ribosome Quality Control Mitigates Proteotoxic Stress in Aneuploid Cells

doi: 10.64898/2026.01.19.700285

Figure Lengend Snippet: A. Representative immunoblot and replicate quantitation of processed-Keima levels in HCT116 RPS3-Keima cells treated for 24 hours with 125nM, 250nM or 500nM Mps1i or DMSO (Ctrl); vinculin was used as loading control. Mean ± SEM, n =4; Kruskal-Wallis test, followed by Dunn’s multiple comparison test: * indicates p =0.0150. B. Representative immunoblot and replicate quantitation of processed-Keima levels in HCT116 RPL28-Keima cells treated for 24 hours with 125nM, 250nM or 500nM Mps1i or DMSO (Ctrl); vinculin was used as loading control. Mean ± SEM, n =4; Kruskal-Wallis test, followed by Dunn’s multiple comparison test: * indicates p =0.0329 (250nM) or p =0.0329 (500nM). C. Representative immunoblots and replicate quantitation of the HCT116 RPS3-Keima cells upon MAD2 or BUB1 siRNA (or non-targeting siRNA); increased levels of processed-Keima indicates ribosome degradation; MAD2 or BUB1 were blotted as knock-down control. Vinculin and tubulin used as loading control. Mean ± SEM, n =4; one sample and Wilcoxon test (each siRNA vs respective non-targeting siRNA=1): * indicates p =0.0257. D. Representative immunoblots and replicate quantitation of the HCT116 RPL28-Keima cells upon MAD2 or BUB1 siRNA (or non-targeting siRNA); increased levels of processed-Keima indicates ribosome degradation; MAD2 or BUB1 have been blotted as knock-down control. Vinculin and tubulin been used as loading control. Mean ± SEM, n =4; one sample and Wilcoxon test (each siRNA vs respective non-targeting siRNA=1): * indicates p =0.0292. E. Representative live-cell images and replicate quantitation of indicated HCT116 Ribo-Keima cells lines treated with Mps1i pulse or DMSO (Ctrl) and collected at 24 hours. Increased Red-Keima intensity indicates ribosome degradation; Hoechst was used to stain DNA; scale bars, 50μm. Upper quartile, lower quartile and median of each violin plot are shown, n =12 fields of view; unpaired Student’s t-test: **** indicates p <0.0001. F. Representative images and replicate quantitation of RFP/GFP ratio in RPE1 RPS3-RFP-GPF cells treated with Mps1i pulse or DMSO (Ctrl) and collected at 24 or 72 hours. RFP/GFP ratio was calculated as explained in Methods and visualised with a rainbow lookup table (calibration bar shown); cyan masks indicate primary nuclei; scale bars 50μm. Upper quartile, lower quartile and median of each violin plot are shown; n=3 biological replicates (30 fields of view each); Kruskal-Wallis test, followed by Dunn’s multiple comparison test: *** indicates p =0.0002, **** indicates p <0.0001.

Article Snippet: The following primary antibodies were used: BUB1 (#ab54893, Abcam), EGFR (in-house polyclonal ab against human EGFR aa 1172–1186, kindly provided by Prof. Di Fiore), GAPDH (#sc-32233, Santa Cruz Biotechnology), HA (#MMS-101P, BioLegend), HSF1 (#4356S, Cell Signaling Technology), Hsp27 (#ADI-SPA-800-D, Enzo Life Science), Hsp70/72 (#ADI-SPA-810-D, Enzo Life Science), Hsp90 (#4877S, Cell Signaling Technology), Keima-red (#M182-3M, MBL), MAD2 (#A300-301A, Bethyl Laboratories), MET (#sc-162, Santa Cruz Biotechnology), mTOR (#2983S, Cell Signaling Technology), PDGFRβ (#3169S, Cell Signaling Technology), Puromycin clone 12D10 (#MABE343, Merck), Tubulin (#T9026, Sigma-Aldrich), Vinculin (#V9131, Sigma-Aldrich), ZNF598 (#ab241092, Abcam).

Techniques: Western Blot, Quantitation Assay, Control, Comparison, Knockdown, Staining

Journal: Molecular Cancer Therapeutics

Article Title: Determinants of Mitotic Catastrophe on Abrogation of the G2 DNA Damage Checkpoint by UCN-01

doi: 10.1158/1535-7163.mct-10-0809

Figure Lengend Snippet: Figure 4. Expression of p31comet attenuates UCN-01–mediated mitotic catastrophe. A, mitosis induced by checkpoint bypass is abolished by p31comet. FLAG- p31comet/HeLa cells were grown in the absence or presence of doxycycline for 30 hours to turn on or off the expression of FLAG-p31comet, respectively. They were treated with IR (16 hours) followed by UCN-01 (12 hours) as indicated. Lysates were prepared and analyzed by immunoblotting. B, p31comet suppresses UCN-01–mediated cell death. Cells were treated as shown in A. Cell viability was determined with trypan blue exclusion analysis (mean SD of 3 experiments). C, expression of MAD2 shRNA or p31comet suppresses mitotic catastrophe. HeLa cells expressing histone H2B-GFP were transfected with control vector (n ¼ 50), plasmids expressing FLAG-p31comet (n ¼ 52), or MAD2 shRNA (n ¼ 74) together with a YFP marker. The cells were irradiated, grown for 16 hours, before treated with UCN-01. Time-lapse microscopy was used to track individual cells for 12 hours. Key and definition are the same as given in Fig. 2A.

Article Snippet: Monoclonal antibodies against b-actin (26), CDK1 (27), cyclin A2 (28), cyclin B1 (22), FLAG (29), a-tubulin (30), and polyclonal antibodies against FLAG (21) were obtained from sources as described previously. www.aacrjournals.org Mol Cancer Ther; 10(5) May 2011 785 D ow nloaded from http://aacrjournals.org/m ct/article-pdf/10/5/784/2320081/784.pdf by guest on 18 July 2024 Polyclonal antibodies against BUBR1 (Bethyl Laboratories), phospho-CDK1Tyr15 (Cell Signaling Technology), phospho-CHK1Ser317 (Abcam), CDC20, phosphohistone H3Ser10 (Santa Cruz Biotechnology), monoclonal antibodies against p31comet (Abcam), CDC27, MAD2, and cleaved PARP(Asp214; BD Biosciences), human nuclear ANA-centromere CREST autoantibody (Fitzgerald Industries), CHK1, cyclin E2, and securin (Santa Cruz Biotechnology) were obtained from the indicated suppliers.

Techniques: Expressing, Western Blot, shRNA, Transfection, Control, Plasmid Preparation, Marker, Irradiation, Time-lapse Microscopy

Journal: Antioxidants

Article Title: Melatonin Alleviates Oxidative Stress Induced by H 2 O 2 in Porcine Trophectoderm Cells

doi: 10.3390/antiox11061047

Figure Lengend Snippet: RT-qPCR measurement of differential gene expression.

Article Snippet: Experiments were conducted using the following primary antibodies: WFDC1 (1:500, OM117200, omnimAbs), HMOX1 (1:500, 27282-1-AP, Proteintech), MXI1 (1:500, OM105330, omnimAbs), PLAG1 (1:500, OM108610, omnimAbs), ViIM (1:200, OM115585, SANTA CRUZ), EGR3 (1:500, OM106724, omnimAbs) and β-actin (1:5000, 66009-1-Ig, Proteintech).

Techniques: Expressing

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Scores of the signature genes resulted from the differential expression analysis using as input the EGA RNA-seq data compared to the three lung samples.

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Expressing

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A The blot of siRNA BAG2 and MDK, C the blot of siRNA MAD2L1. β-Tubulin was used as a loading control and GAPDH was the positive control of the siRNA transfection. As shown in the blots and also in graphs ( B ) and ( D ) there was a significant reduction of the protein level in the silenced cells, as compared to the negative CTRL. E Graph showing the comparison of protein expression in MeT-5A and MSTO cell lines of BAG2, MAD2L1, and MDK (Mann–Whitney test; p value < 0.05 were considered significant, p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****)).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Control, Positive Control, Transfection, Comparison, Expressing, MANN-WHITNEY

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A AOC of MeT-5A and MSTO at 72 h after treatment with siRNA-negative-control (siRNA CTRL−), siRNA BAG2, siRNA MAD2L1, siRNA MDK. The AOC is normalized to the time of transfection ( t 0 ). B Overall, FI of MeT-5A and MSTO was measured at 72 h after silencing; the graphs show the comparison between MeT-5A and MSTO for the three-siRNA transfection. ANOVA test; p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****). C – E End point of apoptosis analysis in MSTO and MeT-5A cells in the presence of 5 µM caspase 3/7 Green Dye and siRNA BAG2, siRNA MAD2L1, and siRNA MDK, respectively. Mann–Whitney test; p value 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Negative Control, Transfection, Comparison, MANN-WHITNEY

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A – D Representative BAG2 immunostainings in RMP, EMM, BMM, and SMM, respectively. Moderate to strong expression in the three types of MPM, while no expression is visible in the hyperplastic mesothelium in RMP (red arrow). E – H Representative MAD2L1 immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is present in the three types of MPM, but there are areas of discernable expression in the mesothelial cells on the pleural surface in RMP as well (blue arrow positive mesothelial area, red arrow negative area). I – L Representative MDK immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is seen in the three types of MPM; however, some focal expression is also detectable in the mesothelial cells on the pleural surface in RMP (blue arrow focal staining, red arrow negative staining). Magnification: RMPs ×400, MPMs ×200.

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Expressing, Staining, Negative Staining

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Graphs for BAG2 immunostaining in RMP vs MPM regardless of subtype ( A ) and RMP vs the different subtypes of MPM ( B ), respectively. Graphs for MAD2L1 immunostaining in RMP vs MPM regardless of subtype ( C ) and RMP vs the different subtypes of MPM ( D ), respectively. Graphs for MDK immunostaining in RMP vs MPM regardless of subtype ( E ) and RMP vs the different subtypes of MPM ( F ), respectively. Kruskal–Wallis test followed by a post hoc Dunn’s test with Bonferroni correction was used to evaluate the statistical difference between the median of each group, p value <0.001(***), <0.01(**), <0.05(*).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Immunostaining