Journal: Neoplasia (New York, N.Y.)

Article Title: A Preclinical Study Combining the DNA Repair Inhibitor Dbait with Radiotherapy for the Treatment of Melanoma 1

doi: 10.1016/j.neo.2014.08.008

Figure Lengend Snippet: Effect of Dbait on H2AX phosphorylation. (A) SK28 and 501mel melanoma cells were transfected with an inactive control oligonucleotide or Dbait ± NU7026 (DNA-PK inhibitor). Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized. Dbait treatment led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. This activity was dependent on DNA-PK activation. Bar, 50 μm. (B) SK28 melanoma cells were transfected with an inactive control oligonucleotide or Dbait. Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized immediately after irradiation and/or Dbait treatment. Irradiation alone resulted in localized γ-H2AX foci representing radio-induced DNA DSBs; Dbait treatment with or without irradiation led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. Bar, 30 μm. (C) SK28 cells were transduced with lentiviruses that express either control, non-targeting shRNA, or shRNA targeting DNA-PKcs. After Dbait transfection, cells were immunostained with mouse monoclonal anti–DNA-PKcs or anti–γ-H2AX. Dbait activity was not detected in cells transduced with shRNA targeting DNA-PKcs. Bar, 50 μm.

Article Snippet: Subconfluent SK28 cells were transduced with lentiviruses that expressed either the control, non-targeting shRNA (shCTL; sc-108080; Santa Cruz Biotechnology, (Dallas, Texas, USA)), or shRNA targeting DNA-PKcs (shDNA-PK; sc-35200-V; Santa Cruz Biotechnology) at a multiplicity of infection of 3 using polybrene (5 μg/ml).

Techniques: Phospho-proteomics, Transfection, Control, Immunofluorescence, Activity Assay, Activation Assay, Irradiation, Transduction, shRNA

Journal: PLoS Pathogens

Article Title: The herpesvirus accessory protein γ 1 34.5 facilitates viral replication by disabling mitochondrial translocation of RIG-I

doi: 10.1371/journal.ppat.1009446

Figure Lengend Snippet: (A) Validation of RIG-I knockdown in HEL cells. Cell lysates from HELs expressing control shRNA (shCtrl) or RIG-I target shRNA (shRIG-I) were subjected to western blot analysis with anti-RIG-I and β-actin antibodies. (B) Effects of γ 1 34.5 on antiviral gene expression in control or RIG-I knockdown HEL cells. Cells infected with wild type HSV-1 or Δγ 1 34.5 (5 pfu/cell) for 8 h were analyzed for transcript levels of IFN-β, Ifit1, Ifit2, and Ccl5 by quantitative PCR analysis. The data were statistically analyzed by one-way ANOVA (**, P < 0.01) with SD (n = 3). (C) Effects of γ 1 34.5 on IRF3 phosphorylation in shCtrl-transfected HEL or RIG-I knockdown HEL. Cells were infected as described in panel B and processed for Western blot analysis with antibodies against p-IRF3, IRF3, ICP27, γ 1 34.5 and β-actin. The experimental data are representative of results from three independent experiments.

Article Snippet: HEL stably expressed Non-Target shRNA (shCtrl) or RIG-I target shRNA (shRIG-I) were selected with puromycin (sc-205821, Santa Cruz Biotechnology) at the concentration 3μg/ml.

Techniques: Biomarker Discovery, Knockdown, Expressing, Control, shRNA, Western Blot, Gene Expression, Infection, Real-time Polymerase Chain Reaction, Phospho-proteomics, Transfection

Journal: PLoS Pathogens

Article Title: The herpesvirus accessory protein γ 1 34.5 facilitates viral replication by disabling mitochondrial translocation of RIG-I

doi: 10.1371/journal.ppat.1009446

Figure Lengend Snippet: (A) Viral replication in Rig-I +/+ or Rig-I -/- MEFs. Cells were infected with wild-type HSV-1 or the γ 1 34.5 deletion virus (Δγ 1 34.5) at a MOI 0.01. At 48 h postinfection, virus yields were determined on Vero cells by plaque assay. (B) Kinetics of viral growth in Rig-I +/+ or Rig-I -/- MEFs. Viral infection was performed as described for panel (A) and viral yields were measured at the indicated time points. (C) Viral replication in control and RIG-I knockdown human lung fibroblasts cells. shCtrl (control) or shRIG-I (RIG-I knockdown) HEL cells were infected with wild type HSV-1 or Δγ 1 34.5 (0.01 pfu/cell). At 48 h postinfection, virus yields were determined by plaque assay. (D) Kinetics of viral growth in control and RIG-I knockdown cells. Viral infection was performed as described in panel (C) and viral yields were measured at the indicated time points. The data are representative of results from three experiments with triplicate samples. Differences between the selected groups were statistically assessed by one-way ANOVA (A and C) or a two-tailed Student’s t test (B and D) (**, P < 0.01).

Article Snippet: HEL stably expressed Non-Target shRNA (shCtrl) or RIG-I target shRNA (shRIG-I) were selected with puromycin (sc-205821, Santa Cruz Biotechnology) at the concentration 3μg/ml.

Techniques: Infection, Virus, Plaque Assay, Control, Knockdown, Two Tailed Test

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: The relative expression of BCAT1 in Ishikawa and HEC-1A cells. BCAT1 expression in Ishikawa and HEC-1A cells transfected with a blank control, shCtrl and shBCAT1. The y-axis indicates the relative expression of BCAT1 in each group tested using RT-qPCR.

Article Snippet: Short Hairpin RNA (shRNA) - mediated BCAT1 knockdown in HEC-1A and Ishikawa cells To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: A. Heat map showing the changes in the concentrations (Z-score) of 26 amino acids and their derivatives after BCAT1 knockdown in HEC-1A cells. B. Intracellular amino acids and their derivative levels in shCtrl cells and shBCAT1 cells.

Article Snippet: Short Hairpin RNA (shRNA) - mediated BCAT1 knockdown in HEC-1A and Ishikawa cells To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Knockdown

Journal: Experimental Hematology & Oncology

Article Title: Targeting phosphoglycerate dehydrogenase in multiple myeloma

doi: 10.1186/s40164-020-00196-w

Figure Lengend Snippet: PHGDH knockdown potentiates bortezomib and proteasome-resistant cell lines are sensitive to PHGDH inhibition. a The knockdown of PHGDH using shRNA was confirmed via WB. b CTG was used to highlight the dependence of the shPHGDH cells on extracellular serine. The sensitivity to drugs was tested by CTG. c , d INA6 KD cells were treated overnight with carfilzomib and bortezomib, respectively. e , f INA6-res and AMO1-res cell lines, respectively, were treated with the indicated doses of NCT-503 overnight. All the presented graphs and calculated IC50s represent three independent experiments with minimum two replicates. Error bars are ± SEM and * P ≤ 0.05 , ** P ≤ 0.01, *** P ≤ 0.001

Article Snippet: Following the manufacturer’s protocol, INA6 knockdown cells (INA6-KD) were transduced with lentiviral particles containing either non-target control shRNA (shCTR) or shRNA targeting PHGDH (shPHGDH) (Santa Cruz Biotechnology, Dallas, TX, USA; sc-108080 and sc-105011-V).

Techniques: Knockdown, Inhibition, shRNA

Journal: Journal of Clinical Laboratory Analysis

Article Title: A long non‐coding RNA OLBC15 promotes triple‐negative breast cancer progression via enhancing ZNF326 degradation

doi: 10.1002/jcla.23304

Figure Lengend Snippet: OLBC15 promotes TNBC in vitro. A, Overexpression efficiency by lentiviral OLBC15 transfection. B, Knockdown efficiency using shRNA targeting OLBC15. ShOLBC15 displayed higher efficiency and was selected as ShOLBC15. C‐D, Effect of OLBC15 on viability of BT‐549 (C) and MDA‐MB‐231 (D) cells transfected with shRNA scrambled control (ShCtrl), ShOLBC15, lentiviral control (OE‐control), or the lentiviral vector carrying OLBC15 (OE‐OLBC15). E, Effect of OLBC15 on migratory capacity of MDA‐MB‐231 (top) and BT‐549 (bottom) cells. F, Quantification of results in (E). **: P < .01

Article Snippet: The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector.

Techniques: In Vitro, Over Expression, Transfection, shRNA, Plasmid Preparation

Journal: Journal of Clinical Laboratory Analysis

Article Title: A long non‐coding RNA OLBC15 promotes triple‐negative breast cancer progression via enhancing ZNF326 degradation

doi: 10.1002/jcla.23304

Figure Lengend Snippet: OLBC15 destabilizes ZNF326 by increasing ubiquitination. A, ZNF326 expression in MDA‐MB‐231 cells transfected with ShCtrl or two shOLBC15 constructs. B, ZNF326 expression in MDA‐MB‐231 cells with or without OLBC15 silence treated with DMSO (MG132‐) or MG132 (MG132+). C, Ubiquitin ligation of ZNF326 in MDA‐MB‐231 cells with or without OLBC15 depletion expressing full‐length Flag‐tagged ZNF326. D, Relative expression of ZNF326 transcripts with either OLBC15 knockdown or overexpression. E, Migration assays for MDA‐MB‐231 cells with OLBC15 knockdown and/or ZNF326 shRNA. F, Quantification data for cellular migration in (E). G, Efficiency of ZNF326 silence on ZNF326 expression. ShZNF326‐2 showed higher efficiency and was selected as ShZNF326. **: P < .01

Article Snippet: The short hairpin RNA (shRNA) for OLBC15 (ShOLBC15) together with a non‐targeting scrambled control (ShCtrl) was obtained from Biovector.

Techniques: Expressing, Transfection, Construct, Ligation, Over Expression, Migration, shRNA

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: ( A ) SK1 mRNA level (left panel) and enzymatic activity (right panel) were measured in B16F10 cells stably transfected with a control (shCtrl) or SK1 targeted shRNA (shSK1, shSK1#2 or shSK1#3). Data are expressed as fold-change over shCtrl B16F10 cells and are means ± sem of 3–5 independent experiments. ( B ) B16F10 murine melanoma cells (3.10 5 ) were injected in the dermis of C57BL/6 mice. After excision 10 days later, tumors were collected and weighed (left panel). Data are means ± sem ( n = 4 to 5 mice per group). The relationship between SK1 enzymatic activity and tumor weight was evaluated with a Pearson correlation analysis (right panel). ( C ) Cellular lipids were extracted from shCtrl or shSK1 tumors and sphingolipid levels were quantified by LC/MS. Levels of S1P (left panel), total ceramide (middle panel) and individual ceramide species (right panel) were normalized to protein content. Results represent means ± sem of 2 independent experiments. ( D ) Tumor volume was determined at the indicated days in the mice that were implanted with shCtrl or shSK1 B16F10 cells, as described in B. Results are from 2 independent experiments performed with 7 mice per group. Values determined for individual tumors are depicted and horizontal lines correspond to means. For all panels, significant differences were evaluated using Student t test.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Activity Assay, Stable Transfection, Transfection, Control, shRNA, Injection, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: ( A , C ) shCtrl or shSK1 B16F10 murine melanoma cells were injected in C57BL/6 mice. Ten days after injection, mice were sacrificed, tumors were collected, and their leukocyte content was analyzed. Bars represent means ± sem of 4 mice per group. Data are representative of two independent experiments. Significant differences were evaluated using Student t test. (A) Cells were counted and the percentage of CD45 and F4/80 among total cells was determined by flow cytometry. (B) Mice ( n = 5–6/group) bearing shCtrl or shSK1 B16 tumors were treated with control liposomes (L-Control) or clodronate-containing liposomes (L-Clodronate) the day before tumor cell injection and then every 4 days for 2 weeks. Tumor volume was determined 13 days after melanoma cell implantation. Values determined for individual tumors are depicted and horizontal lines correspond to means. Data are representative of two independent experiments. Statistical analysis was performed using the Mann-Whitney U -test. (C) Representative flow cytometry density plots. Values indicate the percentages of MHC-II high CD206 low (left panel), MHC-II low CD206 high (middle panel) and iNOS + (right panel) cells among the F4/80 + cells. ( D) Ten days after injection, tumors were collected for mRNA isolation and analysis. Relative mRNA expression (fold induction relative to shCtrl) is depicted for M2 markers (YM1 and ARG-1) and M1 markers (Il12, Tnfα, Ifnγ, Ccl5, Cxcl9, Cxcl10). Significant differences were evaluated using Student t test. NS : not significant.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Injection, Flow Cytometry, Control, Liposomes, MANN-WHITNEY, Isolation, Expressing

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: ( A ) S1P release from shCtrl or shSK1 B16F10 melanoma cells as determined after the conversion of [ 3 H]sphingosine to [ 3 H]S1P. Concentrations of radiolabeled S1P in the medium are expressed as mean ± sem of three independent experiments. Significant differences were evaluated using Student t test. Transwell migration assays were performed to evaluate migration of BMDM ( B and C ) or THP-1 cells ( E – G ). Data are expressed as percent increase or decrease over migration in serum-free medium, and are means ± sem of 2–4 independent experiments. For panels B, C and E-G, significant differences were evaluated using one-way ANOVA with post hoc Tukey test. (B and C) BMDM were pre-treated (+) or not (−) with 2 μM VPC23019 (VPC) or 5 μM W146 for 1 hour. Then, BMDM were incubated for 5 hours in serum-free medium alone (Medium), the conditioned medium (CM) from shCtrl or shSK1 B16F10 melanoma cells containing or not S1P (B) or in serum-free medium containing S1P at the indicated concentrations (C). ( D ) SK1 mRNA expression (left panel), enzymatic activity (middle panel) and S1P release (right panel) were measured in COLO829 melanoma cells transfected either with an empty vector (Vector) or a plasmid encoding human SK1 (SK1). Data are means ± sem of 3 independent experiments. Significant differences were evaluated using Student t test. E and G, THP-1 cells were pre-treated or not with 2 μM VPC23019, 2 μM FTY720 (FTY) or 5 μM W146 for 1 hour. Then, THP-1 cells were incubated for 6 hours in the CM from control (Vector) or SK1-overexpressing (SK1) COLO829 melanoma cells (E) or in serum-free medium containing S1P at the indicated concentrations (G). F, Melanoma CM-induced cell migration was evaluated, as described in E, on THP-1 cells 48 hours after transfection with control (Ctrl) or S1PR1 siRNA (20 nM). Silencing of S1PR1 in THP-1 cells by siRNA was assessed by Western blot.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Migration, Incubation, Expressing, Activity Assay, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: BMDM were incubated for 24 hours in the conditioned medium (CM) from B16F10 melanoma cells ( A and B ) or serum-free medium containing or not 1 μM S1P ( C ). Relative mRNA expression is depicted for M2 markers ( Mrc1 (CD206), Chi3l3 (YM1) and Arg1 ) and M1 markers ( iNos , Il12 , Tnfα , Ifnγ , Ccl5 , Cxcl9 , Cxcl10 ). Data are plotted against the SK1 activity of B16F10 melanoma cells submitted to distinct treatments. (A) CM was prepared from shCtrl or shSK1 B16F10 melanoma cells treated or not with 3 μM SKI-I for 48 hours. (B) BMDM were pre-treated with 2 μM VPC23019 before incubation with the CM from shCtrl B16F10 melanoma cells. Data are expressed as fold-increase over shCtrl B16F10 cells and are means ± sem of 2–6 independent experiments. (C) Data are expressed as fold-change over migration in serum-free medium, and are means ± sem of 3–4 independent experiments. For all panels, significant differences were evaluated using Student t test.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Incubation, Expressing, Activity Assay, Migration

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: ( A ) Analysis of SK1 and TGF-β1 expression in melanoma tumors was carried out using the TCGA database. ( B ) SK1 enzymatic activity (left), TGF-β1 mRNA expression (middle) and TGF-β1 secreted protein level (right) were measured in shCtrl or shSK1 B16F10 melanoma cells treated or not with 3 μM SKI-I for 48 hours. ( C ) TGF-β1 mRNA level was measured in shCtrl B16F10 melanoma cells pretreated or not with 2 μM VPC23019, 2 μM FTY720 or 10 μM JTE013 and incubated with 1 μM S1P for 24 hours. Data are expressed as fold-increase over shCtrl B16F10 cells and are means ± sem of 2–3 independent experiments. Significant differences were evaluated using Student t test. ( D and E ) BMDM were incubated for 48 hours in the conditioned medium (CM) from shCtrl or shSK1 B16F10 melanoma cells. Relative mRNA level is depicted for M2 markers (CD206, YM1 and Arg-1) and M1 markers (iNos, Il12, Tnfα, Ifnγ, Ccl5, Cxcl9, Cxcl10). D, Recombinant murine TGF-β1 (50 ng/ml) was added to the CM from shSK1 B16F10 melanoma cells before incubation with BMDM. E, Anti-TGF-β1 (1 μg/ml) was added to the CM from shCtrl B16F10 melanoma cells before incubation with BMDM. Results represent means ± sem of 2–3 independent experiments. Significant differences were evaluated using Student t test. ( F ) Mice ( n = 6/group) bearing shCtrl B16 tumors were treated with TGF-b-neutralizing antibody or PBS, one day after tumor cell injection and then three times per week for 2 weeks. Tumor volume was determined 13 days after implantation. Values determined for individual tumors are depicted and horizontal lines correspond to means. Data are representative of two independent experiments. Statistical analysis was performed using the Mann-Whitney U -test.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Expressing, Activity Assay, Incubation, Recombinant, Injection, MANN-WHITNEY

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: The expression of BCAT1 in endometrial lesions (magnification, x400). Positive expression of BCAT1 in (A) normal endometrial tissue, (B) atypical endometrial hyperplasia, (C) endometrioid adenocarcinoma, (D) uterine papillary serous carcinoma, (E) endometrial clear cell carcinoma.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: BCAT1 expression in endometrial tissue

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: Association of BCAT1 expression with clinicopathologic parameters in endometrial cancer

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: The relative expression of BCAT1 in Ishikawa and HEC-1A cells. BCAT1 expression in Ishikawa and HEC-1A cells transfected with a blank control, shCtrl and shBCAT1. The y-axis indicates the relative expression of BCAT1 in each group tested using RT-qPCR.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: Ishikawa and HEC-1A cell proliferation as examined by CCK-8 assay. Down-modulation of BCAT1 gene expression significantly suppressed (A) Ishikawa and (B) HEC-1A cell growth.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: CCK-8 Assay, Gene Expression

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: Inhibition of BCAT1 did not induce apoptosis of endometrial cancer cells. Flow cytometry using Annexin V-APC and 7-AAD staining demonstrated that BCAT1 silencing did not change the apoptosis rate of Ishikawa cells and HEC-1A cells compared with control groups.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Inhibition, Flow Cytometry, Staining, Control

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: A. Heat map showing the changes in the concentrations (Z-score) of 26 amino acids and their derivatives after BCAT1 knockdown in HEC-1A cells. B. Intracellular amino acids and their derivative levels in shCtrl cells and shBCAT1 cells.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Knockdown

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: Normalized intensity of isoleucine and leucine in HEC-1A cells. The intensity of isoleucine and leucine were significantly reduced by BCAT1 knockdown.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Knockdown

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: The intensity of intracellular BCAAs in HEC-1A cells. Intracellular reduction of BCAAs after BCAT1 inhibition.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Inhibition

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: Western blotting for the proteins indicated. pS6K levels decreased after BCAT1 knockdown and treatment with 20 mM Gbp for 20 hours.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Western Blot, Knockdown

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: Western blotting for the proteins indicated. The expression levels of AKT and pAKT were not affected by BCAT1 knockdown.

Article Snippet: To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Western Blot, Expressing, Knockdown

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: Identification and validation of RAB11A as an FGFR3-related gene in NMIBC. ( A ) Differential gene expression analysis of TCGA bladder cancer dataset comparing Ta (non-invasive) versus T1–T4 (invasive) stages. RAB11A was identified as one of the significantly upregulated genes in Ta tumors. ( B ) Kaplan–Meier survival analysis of patients in the TCGA BLCA cohort grouped by high and low RAB11A expression ( n = 406). Higher RAB11A expression is associated with poorer overall survival. ( C , D ) Correlation analysis of RAB11A and RAB11B expression with FGFR3 expression in TCGA BLCA dataset. RAB11A showed a stronger positive correlation with FGFR3 than other Rab GTPases. ( E , F ) Immunohistochemical staining of RAB11A in tumor samples from NMIBC patients collected at Renmin Hospital of Wuhan University ( n = 36), showing elevated expression of RAB11A in Ta-stage tumors. Scale bar = 100 μm. ( G , H ) mRNA and protein levels of RAB11A detected in clinical bladder cancer tissues by qRT-PCR and Western blotting, confirming its upregulation in NMIBC compared to MIBC samples. Statistical analysis was performed using unpaired Student’s t-test. Data are shown as mean ± SD from at least three independent experiments. A p -value < 0.05 was considered statistically significant

Article Snippet: Lentiviral constructs encoding shRNAs targeting SREBF2 , RAB11A, or ZDHHC20 , as well as a non-targeting control (shCTL), were obtained from GeneChem (Shanghai, China).

Techniques: Biomarker Discovery, Gene Expression, Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: RAB11A promotes cell proliferation and survival in FGFR3-high RT4 bladder cancer cells. ( A – B ) Western blotting and qRT-PCR were performed to evaluate the knockdown efficiency of RAB11A in RT4 cells following lentiviral transduction with sh RAB11A or non-targeting control (shCTL). ( C ) Cell proliferation was measured using CCK-8 assay at the indicated time points. ( D , E ) Colony-formation assays were performed in RT4 cells expressing control shRNA (shCTL) or sh RAB11A . Quantification includes colony number (colonies containing ≥ 50 cells), mean colony diameter, and total crystal-violet staining intensity (OD 590 ) measured after dye solubilization. Cells were seeded at 500 cells/well in 6-well plates and cultured for 12 days before fixation and crystal-violet staining. ( F – H ) Apoptosis and necrosis were assessed using Annexin V-FITC/PI double staining followed by flow cytometry. Early and late apoptotic cells were quantified in parallel with necrotic cell populations. Statistical analysis was performed using unpaired Student’s t -test. Data are shown as mean ± SD from at least three independent experiments. A p -value < 0.05 was considered statistically significant

Article Snippet: Lentiviral constructs encoding shRNAs targeting SREBF2 , RAB11A, or ZDHHC20 , as well as a non-targeting control (shCTL), were obtained from GeneChem (Shanghai, China).

Techniques: Western Blot, Quantitative RT-PCR, Knockdown, Transduction, Control, CCK-8 Assay, Expressing, shRNA, Staining, Cell Culture, Double Staining, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: SREBF2 directly regulates RAB11A transcription in bladder cancer. ( A ) In-silico transcription-factor screening: the RAB11A (-2000–0 bp) promoter was queried against JASPAR, PROMO, LASAGNA-Search and hTFtarget; overlapping hits are shown as a Venn diagram. ( B ) Overall-survival plot obtained from GEPIA2 using TCGA-BLCA data set stratified by median SREBF2 mRNA. ( C ) QRT-PCR validation of SREBF2 mRNA in RT4 cells stably transduced with two independent shRNAs versus non-target control, GAPDH reference. ( D , E ) Evaluation of RAB11A after SREBF2 knock-down. QRT-PCR for transcript levels ( D ) and immunoblot for protein levels ( E ), GAPDH loading control. ( F ) Dual-luciferase reporter assay in parental RT4 cells co-transfected with full-length or truncated RAB11A -promoter firefly constructs plus SREBF2 or empty vector; Renilla used for normalisation. ( G ) ChIP-qPCR of endogenous SREBF2 at the RAB11A promoter, IgG and H3K4me3 as negative and positive controls. ( H ) Immunofluorescence assay to detect the expression of RAB11A after knockdown of SREBF2 . Scale bar = 20 μm. Data represent mean ± SD of three biological replicates; statistics were performed by unpaired two-tailed t-test ( C , D ) or one-way ANOVA followed by Tukey’s test ( F , G ). A p -value < 0.05 was considered statistically significant

Article Snippet: Lentiviral constructs encoding shRNAs targeting SREBF2 , RAB11A, or ZDHHC20 , as well as a non-targeting control (shCTL), were obtained from GeneChem (Shanghai, China).

Techniques: In Silico, Quantitative RT-PCR, Biomarker Discovery, Stable Transfection, Transduction, Control, Knockdown, Western Blot, Luciferase, Reporter Assay, Transfection, Construct, Plasmid Preparation, ChIP-qPCR, Immunofluorescence, Expressing, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: Phenylalanine 178 of SREBF2 is indispensable for activating RAB11A transcription and for nucleating nuclear condensates that sequester nascent RAB11A transcripts. ( A ) Immunoblot analysis of SREBF2 -knockdown (KD) RT4 cells re-expressing empty vector (pcDNA), wild-type SREBF2 (pcSREBF2), or the phenylalanine-to-alanine mutant (pcSREBF2-F178A). GAPDH serves as a loading control. ( B ) Dual-luciferase reporter assay using a − 2 kb RAB11A promoter construct (pGRAB-Luc). Co-transfection of wild-type SREBF2 markedly elevates promoter activity, whereas F178A produces only a modest increase; pGL3-Basic without an insert provides the basal reference. Data are mean ± SEM of three biological replicates; one-way ANOVA with Tukey post-hoc test. ( C ) ChIP-qPCR in SREBF2 -KD cells transfected with Flag-tagged constructs (pCSRE, pCSRE-F178A, pCVector) Enrichment of the RAB11A promoter is expressed as percentage of input DNA. IgG serves as the negative IP control. ( D ) RNA fluorescence in situ hybridisation (FISH) combined with confocal imaging. Cy3-labelled probes targeting intron 1 of RAB11A (red) were hybridised to SREBF2 -KD cells expressing RFP-tagged SREBF2 constructs (magenta). Controls include scrambled probe, RNase A pretreatment, empty pTagRFP657 vector, and probe-only conditions to validate specificity. Nuclei are counter-stained with DAPI (blue). Scale bar = 10 μm. Data represent mean ± SEM of three independent biological replicates ( n = 3). One-way ANOVA followed by Tukey’s multiple-comparison test was used for ( B ) and ( C ); unpaired two-tailed Student’s t -tests were used for Manders’ co-localisation coefficients (see Results). A p -value < 0.05 was considered statistically significant

Article Snippet: Lentiviral constructs encoding shRNAs targeting SREBF2 , RAB11A, or ZDHHC20 , as well as a non-targeting control (shCTL), were obtained from GeneChem (Shanghai, China).

Techniques: Western Blot, Knockdown, Expressing, Plasmid Preparation, Mutagenesis, Control, Luciferase, Reporter Assay, Construct, Cotransfection, Activity Assay, ChIP-qPCR, Transfection, Fluorescence, In Situ, Hybridization, Imaging, Staining, Comparison, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: Transcriptome and protein analyses reveal that RAB11A modulates FGFR3 membrane localization but not total expression. ( A ) Differential gene expression was analyzed by RNA sequencing (RNA-seq) of RT4 cells transduced with sh RAB11A or shCTL. Volcano plot was generated to visualize significantly upregulated and downregulated genes (adjusted p < 0.05, |log2FC| > 1). ( B ) Heatmap displaying the top 100 differentially expressed genes between the two groups based on Z-score normalized expression values. ( C ) Gene Ontology (GO) enrichment analysis was performed on downregulated genes using the DAVID database, and significant pathways were plotted based on –log10 adjusted p-values. ( D , E ) Quantitative real-time PCR ( D ) and Western blot ( E ) were conducted to detect FGFR3 mRNA and total protein expression in shCTL and sh RAB11A cells. GAPDH served as internal control. ( F ) Flow cytometry was performed to evaluate surface and total levels of FGFR3 in RT4 cells. Cells were stained with anti-FGFR3 antibody with or without permeabilization. Fluorescence intensity was quantified, and the ratio of membrane-to-total FGFR3 signal was calculated. Statistical analysis was performed using unpaired two-tailed Student’s t -test. Data are presented as mean ± SD from at least three independent experiments. A p -value < 0.05 was considered statistically significant

Article Snippet: Lentiviral constructs encoding shRNAs targeting SREBF2 , RAB11A, or ZDHHC20 , as well as a non-targeting control (shCTL), were obtained from GeneChem (Shanghai, China).

Techniques: Membrane, Expressing, Gene Expression, RNA Sequencing, Transduction, Generated, Real-time Polymerase Chain Reaction, Western Blot, Control, Flow Cytometry, Staining, Fluorescence, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: RAB11A regulates FGFR3 palmitoylation and protein stability through interaction with ZDHHC20. ( A – C ) RT4 cells transduced with sh RAB11A or shCTL were treated with 200 ng/mL cycloheximide (CHX) for the indicated times. Whole-cell lysates were collected and analyzed by western blot using antibodies against phospho-FGFR3 (p-FGFR3), total FGFR3, and GAPDH. Band intensities were quantified using ImageJ. ( D – E ) Acyl–biotin exchange (ABE)–like assay using hydroxylamine (HAM) treatment was performed to detect palmitoylated FGFR3. Calnexin served as a positive control. Palmitoylation ratios were calculated based on densitometric comparison of the HAM (+) and HAM (–) lanes. ( F – G ) Co-immunoprecipitation assays were performed in HEK293T cells co-transfected with HA-ZDHHC20 and either Flag-tagged FGFR3 or Myc-tagged RAB11A. Protein complexes were immunoprecipitated with anti-Flag or anti-HA antibodies and analyzed by western blot. ( H ) RT4 cells stably expressing shRNA targeting ZDHHC20 (sh ZDHHC20 ) or control shRNA (shCTL) were subjected to western blot analysis with antibodies against FGFR3, ZDHHC20, and GAPDH. Knockdown of ZDHHC20 markedly reduced FGFR3 protein levels. ( I ) Palmitoylation of FGFR3 was assessed by HAM-dependent ABE assay in ZDHHC20 -knockdown (sh ZDHHC20 ) or control (shCTL) RT4 cells. Band intensities were quantified, showing reduced FGFR3 palmitoylation upon ZDHHC20 depletion. ( J ) Immunofluorescence confocal microscopy of RT4 cells transduced with shCTL, sh RAB11A , or sh ZDHHC20 . Cells were stained for endogenous RAB11A (green), ZDHHC20 (red), and FGFR3 (magenta). Nuclei were counterstained with DAPI (blue). Scale bar = 10 μm. Statistical analysis was performed using unpaired two-tailed Student’s t -test. Data are shown as mean ± SD of three independent experiments. A p -value < 0.05 was considered statistically significant

Article Snippet: Lentiviral constructs encoding shRNAs targeting SREBF2 , RAB11A, or ZDHHC20 , as well as a non-targeting control (shCTL), were obtained from GeneChem (Shanghai, China).

Techniques: Transduction, Western Blot, Positive Control, Comparison, Immunoprecipitation, Transfection, Stable Transfection, Expressing, shRNA, Control, Knockdown, Immunofluorescence, Confocal Microscopy, Staining, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: Cholesterol-rich lipid rafts are required for proper membrane localisation of FGFR3. ( A ) Detergent-resistant membrane (DRM) fractionation. RT4 shCTL, sh RAB11A and sh ZDHHC20 cells Lysates were adjusted to 40% sucrose, over-layered with 35%, 30%, 25%, 15% and 5% sucrose steps and centrifuged at 200 000 g for 18 h (SW-41 rotor, 4 °C). Fractions F1–F4 and the detergent-soluble pellet were collected, equalised for protein and analysed by SDS-PAGE with antibodies against FGFR3, Caveolin-1and Transferrin-R. ( B ) Confocal co-localisation of FGFR3 with GM1. Cells were fixed and surface-labelled with Alexa Fluor 555–cholera-toxin B, briefly permeabilized and stained with anti-FGFR3 (1: 200) plus Alexa 488 secondary antibody. Nuclei were counter-stained with DAPI. Scale bar = 10 μm. ( C , D ) Flow-cytometric assessment of surface FGFR3 after acute modulation of plasma-membrane cholesterol. RT4 cells were treated at 37 °C for 30 min with vehicle or 10 mM methyl-β-cyclodextrin (MβCD); a subset of MβCD-treated cells was subsequently incubated with 2 mM MβCD–cholesterol complex for 1 h, and an additional group received 2 mM MβCD–cholesterol alone. The cells were stained with APC-conjugated anti-FGFR3 extracellular antibody together with an APC-matched isotype control; propidium iodide was used to exclude dead cells. ( C ) Representative histogram overlays; ( D ) geometric mean fluorescence intensity (MFI) normalized to the isotype control from three independent experiments

Article Snippet: Lentiviral constructs encoding shRNAs targeting SREBF2 , RAB11A, or ZDHHC20 , as well as a non-targeting control (shCTL), were obtained from GeneChem (Shanghai, China).

Techniques: Membrane, Fractionation, SDS Page, Staining, Clinical Proteomics, Incubation, Control, Fluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: In vivo validation of the RAB11A–ZDHHC20 axis in regulating bladder tumor growth. ( A ) RT4 cells stably expressing shCTL, sh RAB11A , or sh ZDHHC20 were subcutaneously injected into BALB/c nude mice (5 mice/group). Tumor volume was measured every other day using calipers, and calculated as (length × width²)/2. Tumors were harvested on Day 28 and weighed. ( B ) Tumor tissues were fixed in 4% paraformaldehyde and photographed. ( C ) Formalin-fixed paraffin-embedded tumor sections were subjected to multiplex immunofluorescence. ( D ) Multiplex immunohistochemistry (mIHC) of FFPE xenograft sections using Opal™ TSA reagents (Akoya Biosciences) detected RAB11A (Opal 520, red), ZDHHC20 (Opal 570, magenta) and FGFR3 (Opal 650, green); nuclei were counter-stained with DAPI (blue). Images were captured on a Vectra Polaris multispectral scanner and spectrally unmixed with inForm 2.4. Scale bar = 100 μm. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. Data are shown as mean ± SD. p -values < 0.05 were considered statistically significant

Article Snippet: Lentiviral constructs encoding shRNAs targeting SREBF2 , RAB11A, or ZDHHC20 , as well as a non-targeting control (shCTL), were obtained from GeneChem (Shanghai, China).

Techniques: In Vivo, Biomarker Discovery, Stable Transfection, Expressing, Injection, Formalin-fixed Paraffin-Embedded, Multiplex Assay, Immunofluorescence, Immunohistochemistry, Staining