Journal: EMBO Molecular Medicine

Article Title: De novo pyrimidine synthesis is a collateral metabolic vulnerability in NF2 -deficient mesothelioma

doi: 10.1038/s44321-025-00278-4

Figure Lengend Snippet: ( A ) Cell viability of H2452 NF2 wild-type (sgCtrl) and NF2 knockout (sg NF2 -1, sg NF2 -2) cells treated with various doses of the IMPDH inhibitor for 96 h. Representative data from three independent experiments are shown. The data are presented as the mean ± SD. Two-way ANOVA with multiple comparisons was used for statistical analysis. ( B ) Cell viability of H2452 NF2 wild-type (sgCtrl) and NF2 knockout (sg NF2 -1, sg NF2 -2) cells cultured with the indicated concentrations of glucose. The data are presented as the mean ± SD ( n = 3). Two-way ANOVA with multiple comparisons was used for statistical analysis. ( C–E ) Cell viability of H2452 NF2 wild-type (sgCtrl) and NF2 knockout (sg NF2 -1, sg NF2 -2) cells treated with the indicated doses of the UCK2 inhibitor for 96 h at 10 mM ( C ), 5 mM ( D ) and 2.5 mM ( E ) glucose. Representative data from three independent experiments are shown. The data are presented as the mean ± SD. Two-way ANOVA with multiple comparisons was used for statistical analysis. ( F–H ) Cell viability of H2452 NF2 wild-type (sgCtrl) and NF2 knockout (sg NF2 -1, sg NF2 -2) cells treated with the indicated doses of the DHODH inhibitor for 96 h at 10 mM ( F ), 5 mM ( G ) and 2.5 mM ( H ) glucose. Representative data from three independent experiments were shown. The data are presented as the mean ± SD. Two-way ANOVA with multiple comparisons was used for statistical analysis. ( I ) The differences in LFQ intensities from 480 label-free proteomes of the indicated markers between the H2452 sg NF2 -1 and sgCtrl groups. The data are presented as the mean ± SD ( n = 3). A two-tailed unpaired t test was used for statistical analysis. ( J , K ) The mRNA expression of UPP1 ( J ) and cell viability ( K ) of the indicated cell populations transfected with small interfering RNA (siRNA) targeting the negative control or UPP1 for 72 h. The data are presented as the mean ± SD ( n = 3). Two-way ANOVA with multiple comparisons was used for statistical analysis.

Article Snippet: A UCK2 inhibitor (Catalog #HY-148394) and 15 N-glutamine (Catalog #HY-N0390S) were obtained from MedChemExpress (South Brunswick, NJ, USA).

Techniques: Knock-Out, Cell Culture, Two Tailed Test, Expressing, Transfection, Small Interfering RNA, Negative Control

Journal: Nature Metabolism

Article Title: Salvage of ribose from uridine or RNA supports glycolysis in nutrient-limited conditions

doi: 10.1038/s42255-023-00774-2

Figure Lengend Snippet: ( a ) Protein immunoblot of human THP1 monocytic cells treated with 100 nM PMA alone for 48 h followed by the addition of 100 ng/mL LPS or 1 mg/mL purified yeast RNA for another 48 h and immunoblotted with antibodies to UPP1 and TUBB. Western blot quantification is shown as fold of untreated cells (monocytes). ( b ) Protein immunoblot of human MCSF-matured PBMC treated with 5 µg/ml R848 for 24 h and immunoblotted with antibodies to UPP1 and Actin ( n = 3 donors). Western blot quantification is shown as fold of untreated cells and relative to each donor. ( c ) Expression of UCK1 and UCK2 in THP1 cells as determined by qPCR after treatment as in (a). Data are shown as mean ± SEM with n = 4. ( d ) 13 C 5 -uridine tracer analysis of UMP in THP1 cells treated as in (a) with the exception that glucose-free RPMI media containing 5 mM 13 C 5 -uridine was used. Data are shown as mean ± SEM with n = 4. ( e ) Expression of Upp1 and Il1b in BMDM as determined by qPCR after co-treatment for 24 h with 5 µg/ml R848 and 5 µg/ml BMS-345541, an IKK inhibitor. Data are shown as mean ± SEM with two-sided t-test relative to untreated ( n = 3 mice, P < 1.8 × 10 −2 , P < 4.5 × 10 −2 ). ( f ) 13 C 5 -uridine tracer analysis of citrate and lactate in THP1 cells treated as in (a) with the exception that glucose-free RPMI media containing 5 mM 13 C 5 -uridine was used for the last 6 h. Data are shown as mean ± SEM with two-sided t-test relative to PMA-treated cells with n = 4, P < 4.7 × 10 −4 , P < 1.5 × 10 −2 , P < 1.2 × 10 −2 , P < 2.2 × 10 −2 . ( g ) 13 C 5 -uridine tracer analysis of media lactate in human MCSF-matured PBMC cells treated as in (b) with the exception that glucose-free RPMI media containing 5 mM 13 C 5 -uridine was used was used for the last 6 h. Data are shown as mean ± SEM, with n = 4 donors. TUBB and Actin loading controls were performed on the same gels. All metabolomics included 2 mM (RPMI) or 4 mM (DMEM) L-glutamine and 10% dialyzed FBS.

Article Snippet: RNA was extracted from total cells grown in routine media with a RNeasy kit (Qiagen) and digested with DNase I before murine leukaemia virus reverse transcription using random primers (Promega) and a CFx96 qPCR machine (Bio-Rad) using the following TaqMan assays: Hs01066247_m1 (human UPP1 ), Mm00447676_m1 (mouse Upp1 ), Mm01331071_m1 (mouse Upp2 ), Hs01117294_m1 (human MITF ), Hs01075618_m1 (human UCK1 ), Hs00989900_m1 (human UCK2 ), Mm00550050_m1 (mouse Hmgcs2 ), Hs00427620_m1 (human TBP ) and Mm00782638_s1 (mouse Rplp2 ), Mm00434228_m1 (mouse Il-1B) and Mm01277042_m1 (mouse Tbp).

Techniques: Western Blot, Purification, Expressing

Journal: Oncotarget

Article Title: Teriflunomide restores 5-azacytidine sensitivity via activation of pyrimidine salvage in 5-azacytidine-resistant leukemia cells

doi: 10.18632/oncotarget.19436

Figure Lengend Snippet: Viability of U937 cells and R-U937 cells (A) and of HL-60 cells and R-HL-60 cells (B) after treatment with teriflunomide (TFN) for 72 h. The p -values are indicated. N.S. indicates nonsignificant results of the t -test following two-way ANOVA. The results of three independent experiments are shown. (C) Analysis of EU availability in R-U937 cells (left panel) and R-HL-cells (right panel) without and with 1 μM TFN treatment for 72 h. Green lines: without treatment; red lines: with treatment. (D) Relative mRNA expression levels of UCK1 and UCK2 normalized with the mRNA expression level of ACTB in R-U937 and R-HL-60 cells without and with 1 μM TFN treatment for 72 h. The results of three independent experiments are shown.

Article Snippet: TaqMan gene expression assays (Life Technologies) were used for UCK1 (Hs01075618_m1) and UCK2 (Hs00367072_m1).

Techniques: Expressing

Journal: Cell Death Discovery

Article Title: UCK2 promotes intrahepatic cholangiocarcinoma progression and desensitizes cisplatin treatment by PI3K/AKT/mTOR/autophagic axis

doi: 10.1038/s41420-024-02140-x

Figure Lengend Snippet: A CK2 expression of iCCA tumor ( n = 36) and normal tissue ( n = 8) in TCGA database. B UCK2 expression of iCCA tumor ( n = 30) and normal tissue ( n = 27) in public dataset GSE107943. C UCK2 expression of iCCA tumor ( n = 8) and normal tissue ( n = 8) were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) from SYSUCC Cohort. D UCK2 expression of iCCA tumor ( n = 8) and normal tissue (n = 8) were measured by Western blotting from SYSUCC Cohort. E Representative immunohistochemistry (IHC) staining images of iCCA tumors expressing low or high levels of UCK2. F IHC scores of UCK2 in iCCA tumors and adjacent liver of 70 iCCA patients. G Correlation analysis of UCK2 expression with lymph node metastasis. H Correlation analysis of UCK2 expression with vascular invasion. I Correlation analysis of UCK2 expression with tumor size (≤5 cm vs. >5 cm). J Kaplan–Meier survival curve of overall survival (OS) and disease-free survival (DFS) in 70 iCCA patients, stratified by UCK2 IHC score (UCK2 low expression, n = 40 vs. UCK2 high expression, n = 30). The P value was calculated using the log-rank test. * P < 0.05, ** P < 0.01, *** P < 0.001, according to Student’s t -test.

Article Snippet: The UCK2 overexpression lentiviral vectors and the empty lentiviral vectors were transfected into iCCA cells and designated as RBE/HuCCT1-UCK2 cells and RBE/HuCCT1-Vector cells, while the short hairpin UCK2 lentiviral vectors and the empty lentiviral vectors were also transfected into iCCA cells designated as RBE/HuCCT1-shUCK2 cells and HuCCT1/RBE-Control cells (Shanghai Genechem Co., Ltd., China, customer order number: GOSL0262169).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Cell Death Discovery

Article Title: UCK2 promotes intrahepatic cholangiocarcinoma progression and desensitizes cisplatin treatment by PI3K/AKT/mTOR/autophagic axis

doi: 10.1038/s41420-024-02140-x

Figure Lengend Snippet: A The mRNA level of UCK2 after UCK2 knockdown was confirmed by qRT-PCR. B The protein level of UCK2 after UCK2 knockdown was confirmed by western blotting. C , D Cell growth curve of RBE and HuCCT1 cells transfected with UCK2 shRNA or control. E Colony-forming assays after UCK2 knockdown in HuCCT1 and RBE cells. F Wound-healing assays after UCK2 silencing in HuCCT1 and RBE cells. G Cell migration ability and cell invasion ability after shUCK2-transfection in RBE and HuCCT1 cell. H Xenograft tumors in each group were shown. The mice were sacrificed 32 days post-injection. I Body weight of shUCK2 and Control groups was measured. J Tumor weight of shUCK2 and Control groups was measured. K Tumor growth curves after the injection of shUCK2 and Control RBE cells and Tumor volume was calculated every 4 days. L , M IHC staining of Ki67 in tumors of RBE shUCK2 and Control group. * P < 0.05, **P < 0.01, *** P < 0.001, according to Student’s t -test.

Article Snippet: The UCK2 overexpression lentiviral vectors and the empty lentiviral vectors were transfected into iCCA cells and designated as RBE/HuCCT1-UCK2 cells and RBE/HuCCT1-Vector cells, while the short hairpin UCK2 lentiviral vectors and the empty lentiviral vectors were also transfected into iCCA cells designated as RBE/HuCCT1-shUCK2 cells and HuCCT1/RBE-Control cells (Shanghai Genechem Co., Ltd., China, customer order number: GOSL0262169).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Transfection, shRNA, Control, Migration, Injection, Immunohistochemistry

Journal: Cell Death Discovery

Article Title: UCK2 promotes intrahepatic cholangiocarcinoma progression and desensitizes cisplatin treatment by PI3K/AKT/mTOR/autophagic axis

doi: 10.1038/s41420-024-02140-x

Figure Lengend Snippet: A The mRNA level of UCK2 after UCK2 overexpression was confirmed by qRT-PCR. B The protein level of UCK2 after UCK2 overexpression was confirmed by western blotting. C , D Cell growth curve of RBE and HuCCT1 cells with UCK2 overexpression or control. E Colony-forming assays after UCK2 overexpression in HuCCT1 and RBE cells. F Wound-healing assays after UCK2 overexpression in HuCCT1 and RBE cells. G Cell migration ability and cell invasion ability after UCK2 overexpression in RBE and HuCCT1 cell. H Xenograft tumors in each group were shown. The mice were sacrificed 32 days post-injection. I Body weight and tumor weight of UCK2 overexpression and control groups was measured. Tumor growth curves after the injection of UCK2 overexpression and control RBE cells was calculated every 4 days. J IHC staining of Ki67 in tumors of UCK2 overexpression and Control group. * P < 0.05, ** P < 0.01, *** P < 0.001, according to Student’s t -test.

Article Snippet: The UCK2 overexpression lentiviral vectors and the empty lentiviral vectors were transfected into iCCA cells and designated as RBE/HuCCT1-UCK2 cells and RBE/HuCCT1-Vector cells, while the short hairpin UCK2 lentiviral vectors and the empty lentiviral vectors were also transfected into iCCA cells designated as RBE/HuCCT1-shUCK2 cells and HuCCT1/RBE-Control cells (Shanghai Genechem Co., Ltd., China, customer order number: GOSL0262169).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Control, Migration, Injection, Immunohistochemistry

Journal: Cell Death Discovery

Article Title: UCK2 promotes intrahepatic cholangiocarcinoma progression and desensitizes cisplatin treatment by PI3K/AKT/mTOR/autophagic axis

doi: 10.1038/s41420-024-02140-x

Figure Lengend Snippet: A UCK2 positively correlates with mTOR analyzed by SUSUCC Cohort. B UCK2 positively correlates with mTOR analyzed by GSE107943. C UCK2 positively correlates with AKT and mTOR analyzed by GEPIA2. D The PI3K, phosphorylated AKT (p-AKT), phosphorylated-mTOR (p-mTOR) and AKT and mTOR protein levels after UCK2 knockdown or overexpression. E IHC staining of p-AKT in tumors of each group. F The PI3K, AKT, mTOR, p-AKT and p-mTOR protein levels after UCK2 knockdown or overexpression plus MLN0128. * P < 0.05, ** P < 0.01, *** P < 0.001, according to Student’s t test.

Article Snippet: The UCK2 overexpression lentiviral vectors and the empty lentiviral vectors were transfected into iCCA cells and designated as RBE/HuCCT1-UCK2 cells and RBE/HuCCT1-Vector cells, while the short hairpin UCK2 lentiviral vectors and the empty lentiviral vectors were also transfected into iCCA cells designated as RBE/HuCCT1-shUCK2 cells and HuCCT1/RBE-Control cells (Shanghai Genechem Co., Ltd., China, customer order number: GOSL0262169).

Techniques: Knockdown, Over Expression, Immunohistochemistry

Journal: Cell Death Discovery

Article Title: UCK2 promotes intrahepatic cholangiocarcinoma progression and desensitizes cisplatin treatment by PI3K/AKT/mTOR/autophagic axis

doi: 10.1038/s41420-024-02140-x

Figure Lengend Snippet: A Relative protein levels of LC3II/I, p62 and beclin-1 in RBE and HuCCT1 cells after silencing UCK2 or overexpressing UCK2. B Relative mRNA expression of LC3B and p62 in RBE and HuCCT1 cells after silencing UCK2. C Transmission electron microscopy (TEM) pictures for detecting the autophagosomes RBE and HuCCT1 cells after silencing UCK2 (×2500/3000, 7000/8000 and 20000 magnification). D IHC staining of LC3II in tumors of each group. E Relative protein levels of LC3II/I and p62 in RBE and HuCCT1 cells after silencing UCK2 puls 3-MA. F Relative protein levels of LC3II/I and p62 in RBE and HuCCT1 cells after silencing UCK2 puls Rapamyclin. G Relative protein levels of LC3II/I and p62 in RBE and HuCCT1 cells after UCK2 overexpression puls MLN0128. * P < 0.05, ** P < 0.01, *** P < 0.001, according to Student’s t test.

Article Snippet: The UCK2 overexpression lentiviral vectors and the empty lentiviral vectors were transfected into iCCA cells and designated as RBE/HuCCT1-UCK2 cells and RBE/HuCCT1-Vector cells, while the short hairpin UCK2 lentiviral vectors and the empty lentiviral vectors were also transfected into iCCA cells designated as RBE/HuCCT1-shUCK2 cells and HuCCT1/RBE-Control cells (Shanghai Genechem Co., Ltd., China, customer order number: GOSL0262169).

Techniques: Expressing, Transmission Assay, Electron Microscopy, Immunohistochemistry, Over Expression

Journal: Cell Death Discovery

Article Title: UCK2 promotes intrahepatic cholangiocarcinoma progression and desensitizes cisplatin treatment by PI3K/AKT/mTOR/autophagic axis

doi: 10.1038/s41420-024-02140-x

Figure Lengend Snippet: A Representative immunohistochemistry (IHC) staining images and IHC scores for UCK2 in 10 chemo-resistant and 10 chemo-sensitive iCCA tissues. B Changes of UCK2 expression in both a dose and time manner in RBE and HuCCT1 cells after cisplatin treatment. C Protein expression and mRNA expression of γ-H2AX in shControl or shUCK2-transfected HuCCT1 and RBE cells after treatment with cisplatin (20 or 10 μM, respectively). D Cell viability of shControl or shUCK2-transfected HuCCT1 and RBE cells after treatment with cisplatin (10 or 5 μM, respectively). E Cell viability of Control or UCK2 overexpression transfected HuCCT1 and RBE cells after treatment with cisplatin (20 or 10 μM, respectively). F Protein expression and mRNA expression of γ-H2AX in Control or UCK2 overexpression transfected HuCCT1 and RBE cells after treatment with cisplatin (10 or 5 μM, respectively). * P < 0.05, ** P < 0.01, *** P < 0.001, according to Student’s t -test.

Article Snippet: The UCK2 overexpression lentiviral vectors and the empty lentiviral vectors were transfected into iCCA cells and designated as RBE/HuCCT1-UCK2 cells and RBE/HuCCT1-Vector cells, while the short hairpin UCK2 lentiviral vectors and the empty lentiviral vectors were also transfected into iCCA cells designated as RBE/HuCCT1-shUCK2 cells and HuCCT1/RBE-Control cells (Shanghai Genechem Co., Ltd., China, customer order number: GOSL0262169).

Techniques: Immunohistochemistry, Expressing, Transfection, Control, Over Expression

Journal: Cell Death Discovery

Article Title: UCK2 promotes intrahepatic cholangiocarcinoma progression and desensitizes cisplatin treatment by PI3K/AKT/mTOR/autophagic axis

doi: 10.1038/s41420-024-02140-x

Figure Lengend Snippet: (Right) In iCCA cells, UCK2 overexpression mediated ICC progression through inhibition of autophagy by activating the PI3K/AKT/mTOR signaling pathway and desensitized iCCA to cisplatin treatment via decreasing DNA damage response. (Left) dual inhibition of UCK2 and mTOR suppressed the function of PI3K/AKT/mTOR signaling pathway, then enhance autophagy on iCCA cells resulting in tumor growth inhibition. The schematic diagram was draw by FigDraw (export ID:TPATUf6b66). * P < 0.05, ** P < 0.01, *** P < 0.001, according to Student’s t test.

Article Snippet: The UCK2 overexpression lentiviral vectors and the empty lentiviral vectors were transfected into iCCA cells and designated as RBE/HuCCT1-UCK2 cells and RBE/HuCCT1-Vector cells, while the short hairpin UCK2 lentiviral vectors and the empty lentiviral vectors were also transfected into iCCA cells designated as RBE/HuCCT1-shUCK2 cells and HuCCT1/RBE-Control cells (Shanghai Genechem Co., Ltd., China, customer order number: GOSL0262169).

Techniques: Over Expression, Inhibition