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: Cell Death & Disease

Article Title: PAX5-induced upregulation of IDH1-AS1 promotes tumor growth in prostate cancer by regulating ATG5-mediated autophagy

doi: 10.1038/s41419-019-1932-3

Figure Lengend Snippet: a Top 200 mRNAs that were significantly downregulated in IDH1-AS1-silenced cells were screened out by microarray analysis. b GO analysis of IDH1-AS1-reulated 200 mRNAs. c ATG5 expression level in cells transfected with IDH1-AS1-specific shRNAs and control shRNA was measured using qRT-PCR examination. The level of autophagy-related proteins, LC3 fluorescence intensity and the ratio of autophagosome were detected successively by western blotting ( d ), LC3 immunofluorescence ( e ), and MDC staining ( f ). g ATG5 expression in PCa tissues and its expression association with IDH1-AS1 expression. * P < 0.05, ** P < 0.01. Scale bars for LC3 immunofluorescence and MDC staining equal 200 μm

Article Snippet: To knockdown the expression of PAX5 or IDH1-AS1, the short hairpin RNAs (shRNAs) against PAX5 or IDH1-AS1 (named as shPAX5 or shIDH1-AS1#1/2) and non-specific shRNA (named as shCtrl) were synthesized by GenePharma (Shanghai, China).

Techniques: Microarray, Expressing, Transfection, Control, shRNA, Quantitative RT-PCR, Fluorescence, Western Blot, Immunofluorescence, Staining