Journal: Heliyon

Article Title: Identification of signaling networks associated with lactate modulation of macrophages and dendritic cells

doi: 10.1016/j.heliyon.2025.e42098

Figure Lengend Snippet: Effect of sLA on signaling proteins of interest in DCs and MΦs. ( A ) Schematic experiment was depicted. DCs and MΦs were exposed to either 50 mM sLA or control media, and incubated for 48 h. For Western blot analysis, proteins were separated by size and then transferred to the membrane. Protein detection was done using primary antibodies against several proteins including p-STAT3, p-ERK1/2, p-p38 MAPK, p-STAT1, p-STAT2, p-GSK-3β and β-actin. For flow cytometry, DCs cell suspensions were stained with fluorescently tagged anti-CD11c, p-STAT3, p-ERK and p-p38 MAPK. MΦs were stained with fluorescently tagged anti-F4/80, p-STAT1 and p-GSK-3β. The analysis of the phosphorylated protein expression was performed using a flow cytometer. ( B ), ( D ) Western blot results with represented blots showed the effect of sLA on the levels of p-STAT3, p-ERK1/2 and p-p38 MAPK for DCs. For MΦs, p-STAT1, p-STAT2, p-GSK-3β were shown for the effect of sLA. ( C ), ( E ) Flow cytometric assessment revealed the levels of phosphorylated proteins expression in DCs and MΦs after sLA treatment. The data are demonstrated as the mean ± S.E. from at least three independent experiments, with statistically significant differences calculated by comparing each treatment group to the control. The following symbols indicate the corresponding p-values: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as determined by a two-tailed one-sample t -test.

Article Snippet: The membrane was incubated with each primary antibody targeting phosphorylated STAT1 (p-STAT1, Tyr701, D4A7, Cell signaling technology (CST) #7649), p-STAT2 (Tyr690, CST #4441), p-STAT3 (Ser727, CST #9134), p-SAPK/JNK (Thr183/Tyr185, 81E11, CST #4668), p-SAPK/JNK (Thr183/Tyr185, 98F2, CST #4671), p-NF-κB p65 (Ser536, 93H1, CST #3033), β-Catenin (Ser45, D2U8Y, CST #19807), p-Akt (Ser473, CST #9271), p-Akt (Thr308, D25E6, CST #13038), p-Akt (Ser473, D9E, CST #4060), Akt (pan) (C67E7, CST #4691), p-PTEN (Ser380, CST #9551), p-GSK-3β (Ser9, D85E12, CST #5558), p-c-Raf (Ser259, CST #9421), p-PDK1 (Ser241, C49H2, CST #3438), p-p44/42 MAPK (Erk1/2, Thr202/Tyr204, D13.14.4E, CST #4370), p-p38 MAPK (Thr180/Tyr182, D3F9, CST #4511), and β-actin (CST #4967).

Techniques: Control, Incubation, Western Blot, Membrane, Flow Cytometry, Staining, Expressing, Two Tailed Test

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