Journal: Cancer Biology & Medicine

Article Title: FDX1 expression promotes DSF/Cu-induced cuproptosis in gastric cancer cells

doi: 10.20892/j.issn.2095-3941.2025.0323

Figure Lengend Snippet: Effect of WDR43 knockdown on GC cells. (A) Western blot detection of WDR43 protein expression in GC cells with WDR43 knockdown ( n = 3). (B) Statistical analysis of western blot. (C) CCK-8 detected the proliferation of non-treated GC (CON) and GC cells (siWDR43) after knocking down WDR43 ( n = 3). (D) Invasion assay to detect the invasion ability of cells ( n = 3) (scale bar, 100 μm). (E) Statistical analysis of the results of the invasion experiment ( n = 3). (F) Scratch healing assay to detect cell migration ( n = 3) (scale bar, 500 μm). (G) Statistical analysis of the results of scratch healing experiments ( n = 3). (H) Cell cycle was measured by flow cytometry ( n = 3). (I) Statistical analysis of cell cycle test results ( n = 3). (J) Western blot analysis was performed to detect the levels of CDK2 , cyclinD-1 , and DLAT gene expression in GC cells with FDX1 knockout ( n = 3). (K) Statistical analysis of western blot. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, indicating statistically significant differences.

Article Snippet: The subsequent process included separating the proteins through SDS-PAGE, transferring the proteins onto a PVDF membrane, and combining the membrane with primary antibodies against FDX1, DLAT, WDR43, cyclin dependent-kinase 2 (CDK2), cyclinD-1, and β-actin (Proteintech, Wuhan, China).

Techniques: Knockdown, Western Blot, Expressing, Analysis, CCK-8 Assay, Invasion Assay, Migration, Flow Cytometry, Gene Expression, Knock-Out

Journal: Journal of Translational Medicine

Article Title: Toosendanin suppresses acute myeloid leukemia by targeting DDX5/c-Myc axis to inhibit protein synthesis

doi: 10.1186/s12967-026-08339-z

Figure Lengend Snippet: TSN inhibits AML cell proliferation and induces cell cycle arrest. ( A ) Viability of AML cell lines (MV4-11, MOLM-13, THP-1, and HL-60) treated with TSN for 48 h, as evaluated by CCK-8 assay. Calculated half-maximal inhibitory concentrations (IC₅₀) values are shown. (B, C) Cell cycle distribution of MOLM-13 and MV4-11 cells treated with TSN or DMSO for 48 h, as analyzed by EdU incorporation and flow cytometry. ( B ) Representative flow cytometry plots. ( C ) Quantitative analysis of cells in the G0/G1, S, and G2/M phases ( n = 3). ( D ) Colony-forming capacity of MOLM-13 and MV4-11 cells cultured in methylcellulose medium containing TSN for 8 days. ( E , F ) Western blot analysis of cell cycle regulators CDK1 (34 kDa) and CDK2 (33 kDa) in MOLM-13 and MV4-11 cells after 48 h of TSN treatment ( n = 3). GAPDH (36 kDa) served as the loading control. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Primary antibodies used for western blot analysis included: β-actin (AC004, ABclonal, Wuhan, China; 1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AF1186, Beyotime; 1:1000), β-Tubulin (10068-1-AP, Proteintech, Wuhan, China; 1:1000), CDK1 (A22347, ABclonal; 1:1000), CDK2 (A0294, ABclonal; 1:1000), Caspase-3/Cleaved Caspase-3 (ET1608-64, HUABIO, Hangzhou, China; 1:1000), PARP/Cleaved PARP ( T40050 , Abmart, Shanghai, China; 1:1000), c-Myc ( T55150 , Abmart; 1:1000), and DDX5 (ET1705-32, HUABIO; 1:1000).

Techniques: CCK-8 Assay, Flow Cytometry, Cell Culture, Western Blot, Control

Journal: bioRxiv

Article Title: Non-Genetic Mechanisms of Fractional Resistance to Abemaciclib in Dedifferentiated Liposarcoma

doi: 10.64898/2026.05.22.727236

Figure Lengend Snippet: (A) Experimental workflow of the operating room-to-lab pipeline used for single-cell, multiplexed immunofluorescence profiling. Lipo246 cells were cultured and dedifferentiated primary tumor cells were resected and dissociated to be treated with increasing concentrations of abemaciclib for 24 h. Cells were fixed and subjected to iterative indirect immunofluorescence imaging (4i) consisting of iteration of antibody labeling (block → 1°/2° Ab → elution) and imaging. Images from all 4i rounds are processed through a computational pipeline to perform single-cell segmentation, signal quantification, and feature extraction. (B) Lipo246 and primary tumor cells were stained for 17 cell cycle regulators pRB, RB, Ki-67, CDK2, CDK4, cyclin D1 (cycD1), cyclin E1 (cycE1), Cdt1, E2F1, cyclin A2 (cycA2), cyclin B1 (cycB1), p21, Mdm2, pS6, S6, p53, p16, and integrated DNA. Representative immunofluorescence images from sequential rounds of 4i demonstrate a subset of these regulators for repeated labeling of distinct protein targets within the same cells. Nuclear DNA staining is used for segmentation, image registration, and integration across imaging rounds. (C) Single-cell matrix of 4i-measured cell cycle features. Heatmap of normalized protein expression across all quantified regulators, with individual cells ordered by increasing ratio of phosphorylated to total RB (pRB/RB), computed from 4i measurements. The corresponding single-cell pRB/RB distribution is shown at right and exhibits a bimodal structure used to define low pRB/RB (blue; nonproliferating) and high pRB/RB (pink; proliferating) cell populations. This organization reveals coordinated variation in cell cycle regulators across the pRB/RB-defined axis of cell cycle state.

Article Snippet: To evaluate sensitivity to combined CDK4/6 and CDK2 inhibition, Lipo246 cells were cotreated with abemaciclib (Selleckchem, catalog no. S5716) and the CDK2 inhibitor tagtociclib (MedChem Express, catalog no. HY-137894A).

Techniques: Single Cell, Immunofluorescence, Cell Culture, Imaging, Antibody Labeling, Blocking Assay, Extraction, Staining, Labeling, Expressing

Journal: bioRxiv

Article Title: Non-Genetic Mechanisms of Fractional Resistance to Abemaciclib in Dedifferentiated Liposarcoma

doi: 10.64898/2026.05.22.727236

Figure Lengend Snippet: (A) Distribution of the ratio of phosphorylated to total RB protein (pRB/RB) across individual Lipo246 cells under control (0 nM), 50 nM, and 250 nM abemaciclib treatment. pRB/RB exhibited a bimodal distribution, with a low pRB/RB population corresponding to hypophosphorylated RB and cell cycle arrest, and a high pRB/RB population corresponding to RB hyperphosphorylation and active proliferation. A conservative threshold (vertical red line) was defined to distinguish low pRB/RB and high pRB/RB cells and was used to classify proliferating cells for all subsequent single-cell analyses. (B) 95% CI of proliferating cells for the differences in mean expression (normalized z-scores) between either untreated cells and 50 nM abemaciclib (blue); or untreated and 250 nM abemaciclib (orange). CI overlapping with the dashed line at 0 indicate a lack of statistical significance. (C) Lipo246 cells were quantified, separated into high pRB/RB (pink) or low pRB/RB (Blue) levels. The top panel shows a dose-dependent reduction in cell cycle activity by abemaciclib as demonstrated by a reduction in pRB/RB positivity. While the middle and bottom panel shows an enrichment of CDK2 and cyclin B1 (cycB1) (respectively) in fractionally resistant cells. This enrichment is shown to be statistically significant by ANOVA analysis as seen in the corresponding violin plots. (D) Representative images of the effect of abemaciclib on Lipo246. Cells were treated for 24 hours with 0 (control), 50 nM or 250 nM abemaciclib. Cells were labeled for phosphorylated RB (pRB) which is a marker for actively cycling cells (top panel), cyclin dependent kinase 2 (CDK2)(middle panel) or DAPI for DNA and cycB1 (bottom panel). DAPI staining in the bottom panels shows roughly equal amounts of cells in each drug condition. We can visually observe the quantified depletion of pRB over increasing doses while a fraction of high pRB/RB cells persisting at high drug concentration with of upregulated CDK2 and cycB1.

Article Snippet: To evaluate sensitivity to combined CDK4/6 and CDK2 inhibition, Lipo246 cells were cotreated with abemaciclib (Selleckchem, catalog no. S5716) and the CDK2 inhibitor tagtociclib (MedChem Express, catalog no. HY-137894A).

Techniques: Control, Single Cell, Expressing, Activity Assay, Labeling, Marker, Staining, Concentration Assay

Journal: bioRxiv

Article Title: Non-Genetic Mechanisms of Fractional Resistance to Abemaciclib in Dedifferentiated Liposarcoma

doi: 10.64898/2026.05.22.727236

Figure Lengend Snippet: (A) Distribution of the ratio of phosphorylated to total RB protein (pRB/RB) across individual primary tumor cells under control (0 nM), 50 nM, and 250 nM abemaciclib treatment. A conservative pRB/RB threshold (vertical red line) was applied to identify cells retaining RB phosphorylation and active proliferation under treatment. (B) Differences in mean expression (normalized z-scores) for cell cycle regulators within the high pRB/RB proliferative fraction, comparing untreated cells with 50 nM (blue) or 250 nM (orange) abemaciclib. Points indicate mean differences and error bars denote 95% confidence intervals; intervals overlapping zero indicate nonsignificant differences. (C) Violin plots showing the distributions of selected cell cycle regulators in low pRB/RB (nonproliferating, blue) and high pRB/RB (proliferating, pink) primary tumor cells across treatment conditions. CDK2 and cyclin B1 (cycB1) exhibit selective enrichment within the proliferative fraction under high dose abemaciclib, consistent with fractional resistance. This enrichment is shown to be statistically significant by ANOVA analysis as seen in the corresponding violin plots. D) Representative images of the effect of dedifferentiated liposarcoma primary tumor cells were treated for 24 hours with 0 (control), 50 nM or 250 nM abemaciclib. Cells were labeled for phosphorylated RB (pRB) which is a marker for actively cycling cells (top panel), cyclin dependent kinase 2 (CDK2)(middle panel) or DAPI for DNA and cycB1 (bottom panel). DAPI staining in the bottom panels shows roughly equal amounts of cells in each drug condition. We can visually observe the colocalization of the upregulation of CDK2 and cycB1 in cells expressing pRB at high abemaciclib dose.

Article Snippet: To evaluate sensitivity to combined CDK4/6 and CDK2 inhibition, Lipo246 cells were cotreated with abemaciclib (Selleckchem, catalog no. S5716) and the CDK2 inhibitor tagtociclib (MedChem Express, catalog no. HY-137894A).

Techniques: Control, Phospho-proteomics, Expressing, Labeling, Marker, Staining

Journal: bioRxiv

Article Title: Non-Genetic Mechanisms of Fractional Resistance to Abemaciclib in Dedifferentiated Liposarcoma

doi: 10.64898/2026.05.22.727236

Figure Lengend Snippet: Lipo246 cells were cotreated with the CDK4/6 inhibitor abemaciclib (control, 10 nM, and 50 nM) and a range of CDK2 inhibitor concentrations (0, 0.25, 0.5, 1, 2.5, 5, 10 µM tagtociclib). (A) Proportion of pRB/RB positive cells following treatment with CDK2 inhibitor, tagtociclib. (B) Fold changes in the proportion of pRB/RB positive cells under treatment with tagtociclib. For each abemaciclib condition, fold changes were computed by normalizing the proportion of pRB/RB positive cells to the average proportion of the first three doses of tagtociclib (0, 0.25, and 0.5 µM). Error bars represent the SD across three technical replicates. Dashed lines indicate (A) the proportion or (B) fold change of pRB/RB positive cells without treatment with the CDK2 inhibitor.

Article Snippet: To evaluate sensitivity to combined CDK4/6 and CDK2 inhibition, Lipo246 cells were cotreated with abemaciclib (Selleckchem, catalog no. S5716) and the CDK2 inhibitor tagtociclib (MedChem Express, catalog no. HY-137894A).

Techniques: Control