Journal: Scientific Reports

Article Title: Androgen receptor is a potential novel prognostic marker and oncogenic target in osteosarcoma with dependence on CDK11

doi: 10.1038/srep43941

Figure Lengend Snippet: ( A ) Heatmap of gene probes significantly perturbed in KHOS and U-2OS upon CDK11 knockdown. ( B ) Venn diagram showing the gene number breakdown of differentially expressed genes. ( C ) Heatmap and hierarchal clustering of the common differentially expressed genes in both KHOS and U-2OS. ( D ) Gene network reconstruction utilizing GeneGo. The common differentially expressed genes were used to seed network reconstruction. Two gene networks, both containing CDK11, were merged (green and blue) and trimmed for irrelevant nodes. The genes highlighted in a red shadow were perturbed upon CDK11 knockdown. The Androgen Receptor (AR) is highlighted as a central node and potentially a significant player.

Article Snippet: Wild type CDK11 in pCMV6 vector (RC216465) was purchased from Origene (Rockville, MD). pEGFP-N3 plasmid was purchased from Clontech (Mountain View, CA) and served as control.

Techniques: Knockdown

Journal: Scientific Reports

Article Title: Androgen receptor is a potential novel prognostic marker and oncogenic target in osteosarcoma with dependence on CDK11

doi: 10.1038/srep43941

Figure Lengend Snippet: ( A ) Expressions of CDK11 and AR in osteosarcoma tissues. ( B ) Expressions of CDK11 and AR in osteosarcoma cell lines and normal osteoblast cell lines. ( C ) Expression of AR in osteosarcoma with CDK11 siRNA. ( D ) Representative images of different immunohistochemical staining intensities of AR and CDK11 are shown in osteosarcoma tissues. The percentage of cells showing positive nuclear staining for AR and CDK11 was calculated by reviewing the entire spot. On the basis of the percentage of cells with positive nuclear staining, the staining patterns were categorized into 6 groups: 0, no nuclear staining; 1, 1+, <10% of cells stained positive; 2, 2+, 10% to 25% positive cells; 3, 3+, 26% to 50% positive cells; 4, 4+, 51% to 75% positive cells; and 5, 5+, >75% positive cells (Original magnification, ×400). ( E ) Kaplan-Meier survival curve of patients with osteosarcoma were subgrouped as either CDK11 low staining (staining ≤2) or high staining (staining ≥3). ( F ) Kaplan-Meier disease free survival curve of patients with osteosarcoma were subgrouped as either AR low staining (AR staining ≤2) or high staining (AR staining ≥3).

Article Snippet: Wild type CDK11 in pCMV6 vector (RC216465) was purchased from Origene (Rockville, MD). pEGFP-N3 plasmid was purchased from Clontech (Mountain View, CA) and served as control.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Androgen receptor is a potential novel prognostic marker and oncogenic target in osteosarcoma with dependence on CDK11

doi: 10.1038/srep43941

Figure Lengend Snippet: ( A,B ) U-2OS or KHOS cells were cotransfected with CDK11 (20 ng, 40 ng) and the GoClone promoter reporter vector against the AR promoter (50 ng). CDK11 siRNA (10 nM, 20 nM) was transfected 24 hours prior to transfection with the reporter. 20 ng of an empty luciferase reporter vector was transfected to serve as a baseline, Luciferase activity was measured and normalized to the empty Luc luciferase activity. * P < 0.05, ** P < 0.01 (compared with only AR LUC group).

Article Snippet: Wild type CDK11 in pCMV6 vector (RC216465) was purchased from Origene (Rockville, MD). pEGFP-N3 plasmid was purchased from Clontech (Mountain View, CA) and served as control.

Techniques: Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Induction of beige‐like adipocyte markers and functions in 3T3‐L1 cells by Clk1 and PKCβII inhibitory molecules

doi: 10.1111/jcmm.17345

Figure Lengend Snippet: SiRNA reduction of Clk 1, 2 and 4 levels by specific siRNA reveals that Clk1 inhibition regulates levels of UCP1 and PGC1α. (A) siRNA to Clk1 reduced Clk1 levels by 50% while UCP1 mRNA levels increased fourfold, and PGC1α increased 3.5‐fold. (B) siRNA to Clk2 reduced Clk2 levels by 40%, but only increased UCP1 mRNA by 20% while PGC1α was only increased 50%. (C) siRNA to Clk4 reduced Clk4 by 70%; it reduced UCP1 mRNA by 70%, and PGC1α was increased only 15%. A–C. TG003 (50 nM) treatment consistently increased UCP1 by 3–4 fold, PGC1α by twofold, and had minimal effects on lowering Clk levels. (** p < 0.01 for siClk1,2 and 4 or TG003 vs. scrambled siRNA which was equivalent to control in all cases as evaluated using ANOVA). (D) Oxygen Consumption Rates (OCR) for untreated and TG003‐treated 3T3‐L1 adipocytes. Basal OCR increased 25 pmol/min in TG003‐treated cells vs. control. Proton leak was increased 2.3‐fold with TG003 treatment. Spare receptor capacity increased 50 pmol/min while ATP production was reduced approximately 45% in TG003‐treated cells. Data shown are mean ± SEM (** p < 0.01 for 5 separate experiments performed in duplicate and analysed by Friedman's paired t ‐test)

Article Snippet: Clk1 siRNAs (IDs: SR300856C), Clk2 siRNAs (IDs:SR416008A‐C) and Clk4 siRNAs (IDs: SR408785A‐C) along with scrambled control siRNA was purchased from Origene, and transfected using Origene's siTran transfection reagent.

Techniques: Inhibition, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Induction of beige‐like adipocyte markers and functions in 3T3‐L1 cells by Clk1 and PKCβII inhibitory molecules

doi: 10.1111/jcmm.17345

Figure Lengend Snippet: Binding of TG003 and PGC1α to Clk 1. (A) Steady state SPR of ~1800 RU Clk1 affixed to an Ni‐NTA chip and titrated with TG003 in HBS‐N with 1% DMSO plotted in GraphPad Prism using an one‐site specific binding model with a K D of 25.24 with a 95% confidence interval of 12.93 to 48.53 nM. (B) Steady state SPR of ~400 RU Clk1, ~1000 RU of Clk4 and ~1000 RU Clk2 attached to a CM5 chip via NHS/EDC chemistry indicating binding of Clk1 and Clk4 to TG003 greater than binding of Clk2 to TG003. (C) Steady state SPR of Clk1, Clk2 and Clk4 as in 7B, indicating affinity of Clk1 to PGC1α of 240 nM +/− 28 nM, Clk2 to PGC1α of 980 nM +/− 89 nM, and Clk4 to PGC1α of 509 nM +/− 73 nM. (D) Ligand interaction diagram of TG003 ⁵ bound to Clk1 (PDB ID: 1Z57) generating grid from ligand in crystal structure. (E) 100 ns simulation of Clk1 in the absence of ligand prepared using Schrodinger Protein Preparation Wizard on PDB ID: 1Z57 and NAMD run using CHARMM‐GUI Solution Builder with RMSD of aligned backbone taken over the 1000 frames of the trajectory with each frame equal to 100 ps. (F) ClusPro predicted binding of PGC1α fragment from (PDB ID: 5UNJ) minimized with Protein Preparation Wizard and bound to receptor Clk1 from 7D aligned and one CLK deleted indicating proximal binding to nearby region of Clk1. (G) Heatmap to visualize inhibition of kinases by TG003

Article Snippet: Clk1 siRNAs (IDs: SR300856C), Clk2 siRNAs (IDs:SR416008A‐C) and Clk4 siRNAs (IDs: SR408785A‐C) along with scrambled control siRNA was purchased from Origene, and transfected using Origene's siTran transfection reagent.

Techniques: Binding Assay, Inhibition

Journal: Cell Death & Disease

Article Title: Cytoskeletal protein KRT14 governs cisplatin resistance by modulating eIF4H-dependent ACOX2 translation and lipid metabolism in bladder cancer

doi: 10.1038/s41419-025-08369-3

Figure Lengend Snippet: A Coomassie blue stain comparing lysates (NC vs. KRT14 OE 5637- CR cells). Arrow ~KRT14. B , C Reciprocal endogenous Co-IP in resistant cells confirms KRT14–eIF4H interaction. WB for indicated proteins. D Reciprocal Co-IP confirms interaction of co-expressed Flag-KRT14 and HA-eIF4H. E In vitro GST pull-down showing HA-eIF4H binds GST-KRT14. WB for HA/GST. F Co-IP (eIF4H-IP) shows KRT14 OE enhances eIF4H interaction with eIF4G/E in resistant cells. G WB shows KRT14 OE increases ACOX2 protein, effect attenuated by shEIF4H. H Polysome profiling shows KRT14 OE shifts ACOX2 mRNA to heavier polysomes, partially reversed by shEIF4H. Quantification of mRNA distribution (%). I Dual-luciferase assay (ACOX2 5′UTR reporter) shows KRT14 stimulates activity via eIF4H. RIP-qPCR shows KRT14 OE increases ACOX2 mRNA association with KRT14 ( J ) and eIF4H ( K ). L RNA pull-down using biotinylated ACOX2 5′UTR sense probe captures KRT14 and eIF4H from lysate.

Article Snippet: Human ACOX2 cDNA (Origene RC202901) was inserted into pCDH-CMV-MCS-EF1-Neo with a C-terminal HA tag, and human eIF4H cDNA (Origene RC201007) was cloned into pCMV-HA (Clontech).

Techniques: Staining, Co-Immunoprecipitation Assay, In Vitro, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: Cytoskeletal protein KRT14 governs cisplatin resistance by modulating eIF4H-dependent ACOX2 translation and lipid metabolism in bladder cancer

doi: 10.1038/s41419-025-08369-3

Figure Lengend Snippet: A Schematic of KRT14-FL and truncated domains (Head, Rod, Tail). B GST pull-down shows HA-eIF4H binds KRT14-FL and KRT14-Head fragments in vitro. C Co-IP shows HA-eIF4H co-precipitates with Flag-KRT14-FL and -Head fragments in cells. D Co-IP shows endogenous eIF4H fails to co-precipitate with Flag-KRT14-ΔHead. E – H Functional comparison of KRT14-WT (FL) vs. KRT14-MUT (ΔHead) in resistant cells. E Co-IP (eIF4H-IP) shows MUT fails to enhance eIF4H-eIF4G/E interaction. F Polysome profiling shows MUT fails to enhance ACOX2 mRNA polysome loading. G RIP-qPCR shows MUT fails to enhance eIF4H binding to ACOX2 mRNA. H Cisplatin viability curves (CCK-8, 48 h) show MUT fails to confer resistance.

Article Snippet: Human ACOX2 cDNA (Origene RC202901) was inserted into pCDH-CMV-MCS-EF1-Neo with a C-terminal HA tag, and human eIF4H cDNA (Origene RC201007) was cloned into pCMV-HA (Clontech).

Techniques: In Vitro, Co-Immunoprecipitation Assay, Functional Assay, Comparison, Binding Assay, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: Cytoskeletal protein KRT14 governs cisplatin resistance by modulating eIF4H-dependent ACOX2 translation and lipid metabolism in bladder cancer

doi: 10.1038/s41419-025-08369-3

Figure Lengend Snippet: A – G KRT14 overexpression promotes in vivo tumor growth and confers resistance to cisplatin in a T24 xenograft model. A Representative images of excised tumors, B final tumor weights, and C tumor growth curves consistently show that KRT14 overexpression accelerates tumor growth and mitigates the suppressive effects of cisplatin treatment. At the molecular level, D immunohistochemistry and E Western blot analyses confirm KRT14 overexpression and demonstrate a corresponding increase in the proliferation marker Ki67, as well as ACOX2 and EIF4H. Furthermore, KRT14 overexpression resulted in significantly elevated intratumoral levels of F total cholesterol and G triglycerides. (n = 4); H – L to validate these findings in a more physiologically relevant setting, an orthotopic bladder cancer model was established using the same cell groups. H Representative bioluminescence images and I quantification of total tumor burden demonstrate that KRT14 promotes tumor growth and confers resistance to cisplatin. J Representative gross images of lungs showing metastatic nodules (red arrows), K corresponding H&E staining of lung sections confirming metastatic lesions, and L quantification of pulmonary nodules further reveal that KRT14 potently drives metastatic dissemination and blunts the anti-metastatic effect of cisplatin. Data are presented as mean ± SD (n = 5); p-values are as indicated.

Article Snippet: Human ACOX2 cDNA (Origene RC202901) was inserted into pCDH-CMV-MCS-EF1-Neo with a C-terminal HA tag, and human eIF4H cDNA (Origene RC201007) was cloned into pCMV-HA (Clontech).

Techniques: Over Expression, In Vivo, Immunohistochemistry, Western Blot, Marker, Staining

Journal: Cell Death & Disease

Article Title: Cytoskeletal protein KRT14 governs cisplatin resistance by modulating eIF4H-dependent ACOX2 translation and lipid metabolism in bladder cancer

doi: 10.1038/s41419-025-08369-3

Figure Lengend Snippet: Clinical relevance and prognostic value of KRT14 and ACOX2 in bladder cancer. Violin plots show significantly higher expression of A KRT14 (p < 0.01) and C ACOX2 (p < 0.05) in tumor tissues compared to normal tissues. Kaplan–Meier survival analysis for the patient cohort. High expression of B KRT14 (p = 0.0011) and D ACOX2 (p = 0.0079) was significantly associated with poorer overall survival. E Representative immunohistochemistry images illustrate stronger staining for KRT14 and ACOX2 in various tumor samples (T1–T4) compared to normal tissue (N) at both 200× and 400× magnification. F A scatter plot reveals a significant positive correlation between the expression of KRT14 and ACOX2 in tumors (R = 0.21, p = 0.042). G Representative CT scans from six patients undergoing cisplatin-based chemotherapy, stratified by KRT14 expression status. Cases 1–3, with high KRT14 expression, show progressive or stable disease after treatment. In contrast, Cases 4–6, with low KRT14 expression, show a favorable partial response with significant tumor shrinkage. Red arrows indicate representative tumor lesions. H Schematic model illustrating the proposed mechanism by which KRT14 interacts with eIF4H to selectively promote ACOX2 translation, leading to altered lipid metabolism and ultimately conferring cisplatin resistance and promoting malignant phenotypes in bladder cancer.

Article Snippet: Human ACOX2 cDNA (Origene RC202901) was inserted into pCDH-CMV-MCS-EF1-Neo with a C-terminal HA tag, and human eIF4H cDNA (Origene RC201007) was cloned into pCMV-HA (Clontech).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Nature microbiology

Article Title: Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors

doi: 10.1038/s41564-020-0745-6

Figure Lengend Snippet: (a) Localization of kinetochore protein KKT2 after CLK1 inhibition by AB1. Parasites were incubated or not for 24 h with 2x EC 50 (upper panel) or 6 h with 5 x EC 50 AB1 (lower panel). Representative fluorescence micrographs, showing bloodstream form parasites endogenously expressing N-terminal mNeonGreen (mNG) tagged KKT2. Cells in metaphase and anaphase are shown. Cells were counterstained with DAPI to visualize DNA (cyan). The right panel shows the Nomarsky (DIC) corresponding images. Scale bar, 2μm. Upper right panel shows cell cycle progression after treatment with 2x EC 50 AB1 for 72 h. Data are representative from one of three independent biological replicates with similar results. (b) Localization of KKT2 after CLK1 depletion by RNAi. Representative fluorescence micrographs, showing 24 h induction of CLK1 RNAi in bloodstream form parasites endogenously expressing N-terminal mNeonGreen (mNG) labelled KKT2, compared with not induced control cells in metaphase and anaphase are shown. Cells were counterstained with DAPI to visualize DNA (cyan). The right panel shows the Nomarsky (DIC) corresponding images. Scale bar, 2μm. Data are representative from one of three independent biological replicates with similar results.

Article Snippet: Human CLK1 (hCLK1; P49759) was obtained from Promega.

Techniques: Inhibition, Incubation, Fluorescence, Expressing, Control