Journal: Science advances

Article Title: N-MYC impairs innate immune signaling in high-grade serous ovarian carcinoma.

doi: 10.1126/sciadv.adj5428

Figure Lengend Snippet: Fig. 3. N-MYC represses IFN type I immune signaling. (A) qRT-PCR of IFN type I (IFNA2 and IFNB1) and III (IFNL1, IFNL2, and IFNL3) ligands in CaOV3 MYCN/GFP and GFP cells treated ± DOX (1 μg/ml) for 72 hours. P values were calculated using an unpaired two-tailed Student’s t test. (B) qRT-PCR of IFNAR1 and IFNLR1 (top) and ISGs (bottom) in CaOV3 wild-type cells transfected with scrambled negative control siRNA or siRNAs specific for IFNAR1 and IFNLR1 for 72 hours. (C) Immunoblot of P-STAT1, STAT1, P-STAT2, STAT2, IRF9, and α-tubulin levels in CaOV3 MYCN/GFP and CaOV3 GFP cells treated ± DOX for 24 and 72 hours. Data are representative of three independent experiments. Paired comparisons are shown in the same color for densitometry fold changes. (D) Immunoblot with the indicated antibodies in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX for 72 hours and then treated with IFNB1 (10 ng/ml) for 2 hours. Nuclear and cytoplasmic fractions were prepared and subjected to Western blot. Data are representative of three independent experiments. (E) ChIP-qPCR analysis of IRF9 binding to the ISRE sequence of the ISG15 promoter in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX for 72 hours and then treated with IFNB1 (50 ng/ml) for 30 min. P values were calculated using one-way analysis of variance (ANOVA) with Tukey posttest for pairwise comparison. (F) qRT-PCR of multiple ISGs in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX (1 μg/ml) for 72 hours followed by IFNB1 pulse (10 ng/ml) for 2 hours and then 24-hour chase. P values were calculated using one-way ANOVA with Tukey posttest for pairwise comparison. Data in (A), (B), (E), and (F) are means ± SEM of n = 3 biological replicates. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001. Densitometry analysis was performed using ImageJ software.

Article Snippet: The following antibodies were used in this study: anti- Hu Fc receptor binding inhibitor (#50- 112- 9053, eBioscience), P- STAT1 (Tyr701) (#9167, Cell Signaling Technology), STAT1 (#14994, Cell Signaling Technology), P- STAT2 (Tyr690) (#4441, Cell Signaling Technology), STAT2 (#72604, Cell Signaling Technology), IRF9 (#76684, Cell Signaling Technology), α- tubulin (#3873, Cell Signaling Technology), DYKDDDDK Tag rabbit monoclonal antibody (mAb) (#14793, Cell Signaling Technology), DYKDDDDK Tag mouse mAb (#8146, Cell Signaling Technology), STING (#13647, Cell Signaling Technology), TATA box–binding protein (#8515, Cell Signaling Technology), CD3- PECy7 (#341111, BD Bioscience Technology), RIG- I (#3743, Cell Signaling Technology), MDA- 5 (#5321, Cell Signaling Technology), P- TBK1 (Ser172) (#5483, Cell Signaling Technology), TBK1 (#3504, Cell Signaling Technology), P- IRF3 (Ser396) (#4947, Cell Signaling Technology), N- MYC (#51705, Cell Signaling Technology), VDAC (#4661, Cell Signaling Technology), MAVS (#24930, Cell Signaling Technology), c- MYC (#5605, Cell Signaling Technology), L- MYC (#76266, Cell Signaling Technology), P- STING (Ser366) (#50907, Cell Signaling Technology), hemagglutinin tag (#3724, Cell Signaling Technology), Myc tag (#2278, Cell Signaling Technology), cGAS (#15102, Cell Signaling Technology), anti- rabbit immunoglobulin G (IgG) (H+L) (DyLight 800 4× polyethylene glycol conjugate) (#5151, Cell Signaling Technology), and anti- mouse IgG (H+L) (DyLight 680 conjugate) (#5470, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Two Tailed Test, Transfection, Negative Control, Western Blot, ChIP-qPCR, Binding Assay, Sequencing, Comparison, Software

Journal: Molecular Therapy Oncolytics

Article Title: Acquired chemoresistance can lead to increased resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus

doi: 10.1016/j.omto.2021.11.019

Figure Lengend Snippet: VSV and antiviral protein expression (A and B) C and GR cells were either mock treated or infected with VSV-ΔM51 at MOIs of 1.0, 0.1, and 0.01. Protein sample were analyzed at 24 h p.i. (A) and 48 h p.i. (B) by western blotting for expression of phospho-STAT1 (p-STAT1), phospho-STAT2 (p-STAT2), STAT1, STAT2, and VSV proteins (G, N/P, and M). Cell line and treatment conditions are indicated above blots. Equal protein loading is indicated by Coomassie blue. (C and D) The effect of ruxolitinib on VSV-ΔM51 replication kinetics based on GFP fluorescence. C and GR cells were either mock treated, treated only with ruxolitinib, treated only with VSV-ΔM51, or treated with both VSV-ΔM51 and ruxolitinib. Cells were infected at MOI of 0.01. Ruxolitinib was added to cells after 1 h VSV incubation period. GFP fluorescence was measured from 1 to 80 h p.i. The data points and error bars shown represent the means and SEM of the means, respectively. Control and GR cell lines 1–3 are combined for each treatment.

Article Snippet: Membranes were then incubated in TBS-T with 5% BSA or milk with 0.02% sodium azide and a 1:5,000 dilution of rabbit polyclonal anti-VSV antibodies (raised against VSV virions), a 1:1,000 dilution of rabbit anti-phospho-STAT1 (catalog number 9177S, clone p-S727, Cell Signaling), a 1:1,000 dilution of rabbit anti-STAT1 (catalog number 14994T, clone D1K9Y, Cell Signaling), a 1:1,000 dilution of rabbit anti-phospho-STAT2 (catalog number 600-401-A93S, clone p-Y689, Rockland), a 1:1,000 dilution of rabbit anti-STAT2 (catalog number 4594, Cell Signaling), a 1:1,000 dilution of rabbit anti-phospho-STAT3 (catalog number 9134P, clone Y705, Cell Signaling), a 1:1,000 dilution of mouse anti-STAT3 (catalog number 9139P, clone 124H6, Cell Signaling), a 1:1,000 dilution of rabbit anti-MX1 (catalog number 13750-1-AP, Proteintech), a 1:1,000 dilution of rabbit anti-MX2 (catalog number 43924S, clone E7Y8H, Cell Signaling), a 1:1,000 dilution of rabbit anti-IFI16 (catalog number 14970S, clone D8B5T, Cell Signaling), a 1:1,000 dilution of rabbit anti-APOBEC3B (catalog number 41494S, clone E9A2G, Cell Signaling), a 1:1,000 dilution of rabbit anti-ISG15 (catalog number 2758S, clone 22D2, Cell Signaling), a 1:1,000 dilution of rabbit anti-CDK14-PFTK1 (catalog number 21612-1-AP, Proteintech), a 1:1,000 dilution of rabbit anti-LARGE2/GYLTL1B (catalog number PA5-63331, Invitrogen), a 1:1,000 dilution of rabbit anti-STING (catalog number 13647S, clone D2P2F, Cell Signaling), a 1:1,000 dilution of rabbit anti-phsopho-TBK1/NAK (catalog number 5483P, clone S172, Cell Signaling), a 1:1,000 dilution of rabbit anti-cGAS (catalog number 79978, clone E5V3W, Cell Signaling), or a 1:1,000 dilution of rabbit anti-cyclin B1 (catalog number 12231T, clone D5C10, Cell Signaling).

Techniques: Expressing, Infection, Western Blot, Fluorescence, Incubation

Journal: Computational and Structural Biotechnology Journal

Article Title: A five-protein prognostic signature with GBP2 functioning in immune cell infiltration of clear cell renal cell carcinoma

doi: 10.1016/j.csbj.2023.04.015

Figure Lengend Snippet: GBP2 enhances metastasis of ccRCC cells through JAK-STAT signaling. A. Western blotting validation of GBP2 expression in ccRCC cells upon knockdown of GBP2. B. Transwell assays were used to test the migration and invasion abilities upon knockdown of GBP2. * *P < 0.01, * **P < 0.001. Scale bar, 600 µm. C. Western blotting validation of GBP2 expression in ccRCC cells transfected with indicated constructs. D. Transwell assays were used to test the migration and invasion abilities of ACHN and 769-P cells transfected with indicated constructs. * *P < 0.01, * **P < 0.001. Scale bar, 600 µm. E-F. Gene Set Enrichment Analysis revealed that high GBP2 expression was mainly enriched in JAK/STAT signaling based on proteome and transcriptome data from CPTAC (E) and TCGA (F). G. Protein-protein interaction network for GBP2 in ccRCC was constructed using GeneMANIA. Different colors of the network edge indicate the bioinformatics methods applied: physical interaction, predicted, and co-expression. H. The correlation of GBP2 with STAT2/STAT3 at both RNA levels (TCGA) and protein levels (CPTAC) in ccRCC was determined. I-J. GBP2 siRNAs (I) or overexpression vectors (J) were transfected into ccRCC cells. The phosphorylation of STAT family proteins was examined by western blotting. K. Transwell assays were used to test the migration abilities of GBP2-expressing ACHN and 769-P cells treated with JAK inhibitor Ruxolitinib. * * P < 0.01, * ** P < 0.001. n.s, no significant. Scale bar, 600 µm.

Article Snippet: STAT1 (Cell Signaling Technology, Boston), STAT2 (Proteintech, Wuhan), STAT3 (Beyotime Biotechnology, Shanghai), Phospho-STAT1 (Tyr701) (Cell Signaling Technology, Boston), Phospho-STAT2 (Tyr690) (Beyotime Biotechnology, Shanghai), Phospho-STAT3 (Tyr705) (Beyotime Biotechnology, Shanghai).

Techniques: Western Blot, Expressing, Migration, Transfection, Construct, Over Expression