Journal: Science Advances

Article Title: GSK3β and Plk1 sequentially phosphorylate ATP–citrate lyase to promote homologous recombination

doi: 10.1126/sciadv.aeg1097

Figure Lengend Snippet: ( A to E ) HeLa cells were transfected with the indicated siRNAs. Forty-eight hours posttransfection, cells were irradiated (2 Gy) and processed for immunoblotting (A) or immunofluorescence staining 4 hours later. Representative images are shown (B). Quantification of the percentage of γH2AX-positive cells with >5 BRCA1 foci (C), BRCA1 foci intensity in γH2AX-positive cells (D), and nuclear γH2AX intensity (E) is shown. Data are presented as means ± SD from three independent experiments [ n > 300 cells per condition for (C) and (E); n > 150 cells per condition for (D)]. ( F to I ) HeLa cells were transfected with the indicated siRNAs. Forty-eight hours posttransfection, cells were irradiated (2 Gy) and processed for immunoblotting (F) or immunofluorescence staining 4 hours later. Representative images are shown (G). Quantification of the percentage of cyclin A2–positive cells with >5 γH2AX foci (H) and nuclear γH2AX intensity in cyclin A2–positive cells (I) is shown. Data are presented as means ± SD from three independent experiments [ n > 300 cells per condition for (H) and (I)]. ( J to L ) HeLa cells were transfected and irradiated as in (F) and processed for immunofluorescence staining 4 hours later. Representative images are shown (J). Quantification of the percentage of cyclin A2–positive cells with >5 BRCA1 foci (K) and BRCA1 foci intensity in cyclin A2–positive cells (L) is shown. Data are presented as means ± SD from three independent experiments [ n > 300 cells per condition for (K); n > 150 cells per condition for (L)]. Scale bars, 10 μm (B, G, and J). A.U., arbitrary units (D, E, I, and L). N.S., not significant. See also fig. S4.

Article Snippet: Primary antibodies used were as follows: ACLY (Abcam, ab40793), Flag tag (GenScript, A01868; for immunoblotting), GST (Sigma-Aldrich, G7781), HA tag (BioLegend, 16B12), Myc-tag (05-724, Millipore), MBP (New England BioLabs, E8032), pan-pSer/Thr (ABclonal, AP0893), pan-Ser-pThr-Pro (Cell Signaling Technology, D73F6; 5243), GSK3α (ABclonal, A19060), GSK3β (ABclonal, A6164), ACLY-pS455 (Cell Signaling Technology, 4331), BRCA1 (Santa Cruz Biotechnology, sc-6954), γH2AX (Abcam, ab81299), cyclin A2 (ABclonal, A19036), cyclin A2 (Proteintech, 66391-1-Ig), 53BP1 (made in-house), pan-AKT (ABclonal, A18120), RAD51 (made in-house), histone H4 (Sigma-Aldrich, 07-108), H4K16ac (Abcam, ab109463), α-tubulin (ABclonal, AC012), GFP (Abmart, M20004), Plk1 (Abcam, ab17057), and Plk1 phospho-T210 (BD Transduction, 558400).

Techniques: Transfection, Irradiation, Western Blot, Immunofluorescence, Staining

Journal: Science Advances

Article Title: GSK3β and Plk1 sequentially phosphorylate ATP–citrate lyase to promote homologous recombination

doi: 10.1126/sciadv.aeg1097

Figure Lengend Snippet: ( A to D ) Parental HeLa cells and HeLa cells stably expressing ACLY-GFP (WT or S442A) were transfected with control or ACLY siRNA. Forty-eight hours posttransfection, cells were irradiated (2 Gy) and processed for immunoblotting (A) or immunofluorescence staining 4 hours later. Representative images are shown (B). Quantification of the percentage of γH2AX-positive cells with >5 BRCA1 foci (C) and BRCA1 foci intensity in γH2AX-positive cells (D) is shown. Data are presented as means ± SD from three independent experiments [ n > 300 cells per condition for (C); n > 150 cells per condition for (D)]. ( E to H ) Parental HeLa cells and HeLa cells stably expressing ACLY-GFP (WT or S442A) were transfected with control or ACLY siRNA. Forty-eight hours posttransfection, cells were irradiated (2 Gy) and processed for immunoblotting (E) or immunofluorescence staining 4 hours later. Representative images are shown (F). Quantification of the percentage of cyclin A2–positive cells with >5 BRCA1 foci (G) and BRCA1 foci intensity in cyclin A2–positive cells (H) is shown. Data are presented as means ± SD from three independent experiments [ n > 300 cells per condition for (G); n > 150 cells per condition for (H)]. Scale bars, 10 μm (B and F). See also figs. S7 and S8.

Article Snippet: Primary antibodies used were as follows: ACLY (Abcam, ab40793), Flag tag (GenScript, A01868; for immunoblotting), GST (Sigma-Aldrich, G7781), HA tag (BioLegend, 16B12), Myc-tag (05-724, Millipore), MBP (New England BioLabs, E8032), pan-pSer/Thr (ABclonal, AP0893), pan-Ser-pThr-Pro (Cell Signaling Technology, D73F6; 5243), GSK3α (ABclonal, A19060), GSK3β (ABclonal, A6164), ACLY-pS455 (Cell Signaling Technology, 4331), BRCA1 (Santa Cruz Biotechnology, sc-6954), γH2AX (Abcam, ab81299), cyclin A2 (ABclonal, A19036), cyclin A2 (Proteintech, 66391-1-Ig), 53BP1 (made in-house), pan-AKT (ABclonal, A18120), RAD51 (made in-house), histone H4 (Sigma-Aldrich, 07-108), H4K16ac (Abcam, ab109463), α-tubulin (ABclonal, AC012), GFP (Abmart, M20004), Plk1 (Abcam, ab17057), and Plk1 phospho-T210 (BD Transduction, 558400).

Techniques: Stable Transfection, Expressing, Transfection, Control, Irradiation, Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Patient-derived organoids guide personalized therapy for KRAS-mutant pancreatic cancer: synergistic MEK/mTOR inhibition and predictive chemotherapy responses

doi: 10.3389/fimmu.2026.1760379

Figure Lengend Snippet: Discovery of synergistic drug combinations that enhance the MEKi therapy. (A) The representative bliss synergy score heatmap of MEKi combination with MTORi is shown. Red, synergy; green, antagonism; white, no effect. The averaged dose–response curves of three independent experiments (with three technical replicates) are shown. Synergy avg , average bliss synergy score. (B) The representative bliss synergy score heatmap of the MEKi combination with Pan-CDKi is shown. Red, synergy; green, antagonism; white, no effect. The averaged dose–response curves of three independent experiments (with three technical replicates) are shown. Synergy avg , average bliss synergy score. (C) Dose–response curves of four PDOs treated with MEKi alone or in combination with pan-CDKi or MTORi for 96 h. Cell viability was measured by using CellTiter-Glo ® 3D Cell Viability Assay. Data are mean ± s.d. (D) Representative images of PDAC organoid 028 treated with the vehicle, trametinib, flavopiridol, AZD8055 alone, or a dual combination for 72 h. (E) PDO028, PDO099, and PDO433-LM were treated with the indicated drugs for 72 h. Cell lysates were immunoblotted to determine the levels of phosphorylated and total MEK, ERK, cyclin A2, cyclin D1, phosphorylated RB, and the loading control GAPDH. (F) PDO028 and PDO433-LM were treated with the indicated drugs for 72 h. The cell lysates were immunoblotted to determine the levels of phosphorylated and total MEK, ERK, S6, phosphorylated RB, and the loading control GAPDH. Error bars indicate mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA test.

Article Snippet: Cyclin A2 rabbit pAb , Abclonal , A7632.

Techniques: Viability Assay, Control

Journal: iScience

Article Title: SKA2 promotes gastric cancer progression by regulating glutathione metabolism

doi: 10.1016/j.isci.2026.115202

Figure Lengend Snippet: Knocking down SKA2 induces gastric cancer cell lines G2/M arrest (A and B) Cell cycle analysis of SKA2 knockdown in SNU638 and NUGC3 cell lines. Percentage of flow cytometric histogram images shows the effects of SKA2. (C) Western blotting analysis of the expression of Cyclin D1, Cyclin A2, Cyclin B1, and SKA2 in SNU638 and NUGC3 SKA2-knockdown cell lines. α-Tubulin was used as the internal control. (D and E) Cell cycle analysis of SKA2 overexpression in SNU638 and NUGC3 SKA2-knockdown cell lines. Percentage of flow cytometric histogram images shows the effects of SKA2. (F) Western blotting analysis of SKA2 overexpression in SNU638 and NUGC3 SKA2-knockdown cell lines using anti-Cyclin D1, anti-Cyclin A2, anti-Cyclin B1, and anti-SKA2 antibodies. α-Tubulin was used as the internal control. Representative flow cytometry histograms and blotting images are shown from 3 biologically independent experiments. Data in (A)–(B) and (D)–(E) are presented as mean ± SD, n = 3 biologically independent experiments. p values are based on a one-way ANOVA test. ns, no significance (∗∗ p < 0.01; ∗∗∗ p < 0.001).

Article Snippet: Cyclin A2 , Cell Signaling Technology , Cat#4656; RRID: AB_2071958.

Techniques: Cell Cycle Assay, Knockdown, Western Blot, Expressing, Control, Over Expression, Flow Cytometry

Journal: iScience

Article Title: SKA2 promotes gastric cancer progression by regulating glutathione metabolism

doi: 10.1016/j.isci.2026.115202

Figure Lengend Snippet: Knocking down SKA2-induced cell-cycle arrest and apoptosis through the SKA2/ROS/ATM axis in GC cell lines (A) Western blotting analysis of γ-H2AX (Ser139), ATM, p -ATM (Ser1981), p -Chk2 (Thr68), and SKA2 expression in SNU638 and NUGC3 SKA2-knockdown cell lines. (B) Western blotting analysis of SKA2 overexpression in SNU638 and NUGC3 SKA2-knockdown cell lines using antibodies against γ-H2AX (Ser139), ATM, p -ATM (Ser1981), p -Chk2 (Thr68), and SKA2. (C) Western blotting analysis of KU-55933 treatment in SNU638 and NUGC3 SKA2-knockdown cell lines using antibodies against cyclin D1, cyclin A2, cyclin B1, p -Chk2 (Thr68), PARP, Cleaved-Caspase3, JNK, p -JNK (Thr183/Tyr185), ATM, p -ATM (Ser1981), and SKA2. (D) Western blotting analysis of BML-277 treatment in SNU638 and NUGC3 SKA2-knockdown cell lines using antibodies against cyclin D1, cyclin A2, cyclin B1, p -Chk2 (Thr68), and SKA2. (E) Western blotting analysis of P38, p-P38 (Thr180/Tyr182), ERK, p -ERK1/2 (Thr202/Tyr204), JNK, p -JNK (Thr183/Tyr185), and SKA2 expression in SNU638 and NUGC3 SKA2-knockdown cell lines. (F) Western blotting analysis of the rescue effect of SKA2 overexpression on MAPK pathway markers (ERK, p -ERK1/2, JNK, and p -JNK) in SNU638 and NUGC3 SKA2-knockdown cell lines. (G) Western blotting analysis of JNK-IN-8 treatment in SNU638 SKA2-knockdown cell lines using antibodies against PARP, cleaved-caspase3, JNK, p -JNK (Thr183/Tyr185), and SKA2. α-Tubulin was used as the internal control for all blots. Representative blotting images are shown from 3 independent experiments.

Article Snippet: Cyclin A2 , Cell Signaling Technology , Cat#4656; RRID: AB_2071958.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Control

Journal: bioRxiv

Article Title: Targeting Distinct Cell Cycle Nodes Overcomes KRAS/RAS Inhibitor Resistance

doi: 10.64898/2026.03.10.710937

Figure Lengend Snippet: (A) GSEA analysis illustrating the differential effect of MRTX849 on the genes associated with E2F target pathway and the proteins associated with G2/M checkpoint in cell cycle machinery. (B) Heat map represents the differential effect of MRTX849 in downregulating the indicated genes and proteins that are involved in cell cycle from MiaPaCa-2-WT and MiaPaca-2-MR cells following 48-hour treatment. (C) Biochemical analysis on the indicated sensitive (WT) and resistance cell lines to compare the effect of MRTX849 on RB phosphorylation and cyclin A expression at different doses after treating the cells for 48 hours. (D) GSEA analysis highlighting pathways associated with genes and proteins that are differentially expressed between MiaPaCa-2-MR and MiaPaCa-2-WT cells. (E) Volcano plots of differentially expressed genes and proteins in MiaPaCa-2-MR versus MiaPaCa-2-WT cells. (F) Heat map illustrating the effects of indicated targeted therapeutic agents on normalized fold change in cell growth in combination with DMSO or mutant-selective KRAS inhibitors. (G) Synergistic interactions between KRAS inhibitors and erlotinib or AZD4547 in AsPC-1-MR, UM53, and MiaPaCa-2-MR cells. Heat maps depict normalized fold change in growth rate following treatment with increasing concentrations of KRAS inhibitors in combination with erlotinib or AZD4547. Bliss synergy scores were calculated using the SynergyFinder online platform.

Article Snippet: Antibodies against Cyclin A (Cat# AF5999) and β-actin (Cat# MAB8929) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Expressing, Mutagenesis