Journal: International Journal of Molecular Sciences

Article Title: IL-8 Secreted from M2 Macrophages Promoted Prostate Tumorigenesis via STAT3/MALAT1 Pathway

doi: 10.3390/ijms20010098

Figure Lengend Snippet: STAT3 was indispensable for IL-8 mediated MALAT1 induction. ( A ) M2 macrophages elevated the phosphorylation level of STAT3 in PCa cell lines. 22Rv1 (left) or LNCaP (right) cell line was co-cultured with M2 macrophages (normal culture was performed as the negative control). After incubation for 8 h, cells were treated with 50 nM IL-8 Ab (IgG was performed as negative control); after further incubation for 36 h, the cells were lysed and the pSTAT3 expression level was detected by western blot assay. GAPDH was applied as loading control. ( B ) rhIL-8 induced phosphorylate of STAT3 in PCa cell lines. 22Rv1 (left) or LNCaP (right) were treated with rhIL-8 (0, 10 and 100 μg/mL) respectively. After incubation for 48 h, the cells were lysed and the expression level of pSTAT3 was measured by western blot. GAPDH was applied as loading control. ( C ) STAT3 inhibitor blocked IL-8 induced MALAT1 expression in PCa cell lines. 22Rv1 (left) or LNCaP (right) cell line was treated with rhIL-8 (0 or 100 μg/mL), after incubation for 8 h, 100 µM S3I-201 (DMSO was performed as negative control) was added; after further incubation for 36 h, PCa cells were harvested for total RNA extraction. The level of MALAT1 mRNA was analyzed by quantitative PCR. ( D ).STAT3 inhibitor blocked M2 macrophages induced MALAT1 expression in PCa cell lines. 22Rv1 (left) or LNCaP (right) cell was co-cultured with M2 macrophages (normal culture was performed as negative control), after incubation for 8 h, 100 µM S3I-201 (DMSO was performed as negative control) was added; after further incubation for 36 h, PCa cells were harvested for total RNA extraction. The level of MALAT1 mRNA was analyzed by quantitative PCR. ( E ) STAT3 induced MALAT1 expression in PCa cell lines. 22Rv1 (left) or LNCaP (right) cell was transfected with pcDNA3.1- STAT3 (0, 500, 1000, and 1500 ng); at 48 h, the PCa cells were harvested for total RNA extraction. The level of MALAT1 mRNA was analyzed by quantitative PCR. All data presented are the mean ± SD (** p < 0.01, *** p < 0.001) of triplicate determination from three independent experiments.

Article Snippet: The putative wild-type and mutated STAT3-binding sites in the MALAT1promoter region were cloned into pGL4.22 luciferase vector (Promega, Madison, WI, USA) to contruct wM-Luc and mM-Luc; phRL-TK were purchased from Promega (Promega, USA); pLKO.1-puro, pVSV-G and pCMV-Δ8/9 were a generous gift from Bob Weinberg (#8453-#8455, Addgene, USA); Dimethyl sulfoxide (DMSO) was purchased from Sigma (Sigma, USA); S3I-201 was purchased from Selleck (Selleck, Houston, TX, USA).

Techniques: Cell Culture, Negative Control, Incubation, Expressing, Western Blot, RNA Extraction, Real-time Polymerase Chain Reaction, Transfection

Journal: Scientific Reports

Article Title: Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin

doi: 10.1038/s41598-018-34938-y

Figure Lengend Snippet: Phosphoarray analysis of signaling responses to sequential treatments. ( A ) HCA7 cells were subjected to different regimens containing 10 μg/ml cetuximab and/or 50 μM oxaliplatin: (1) control medium for 24 hours (white), (2) control medium for 24 hours followed by oxaliplatin for 1 hour (black), (3) cetuximab for 24 hours followed by oxaliplatin for 1 hour (red), or (4) oxaliplatin for 24 hours followed by cetuximab for 1 hour (green). Phosphorylation profiles were analyzed using Proteome Profiler Human Phospho-Kinase Array Kit and quantified by densitometry. Mean +/− range is shown for four selected proteins. All results are shown in Suppl. Fig. . ( B ) HCA7 cells were subjected to different regimens containing or not 10 μg/ml cetuximab and/or 50 μM oxaliplatin as indicated in the figure. The expression or phosphorylation of selected proteins was analyzed by Western blotting. Full-length blots are presented in Supplementary Fig. . ( C ) Apoptosis of HCA7 cells was analyzed by annexin V staining after treating the cells first with or without 200 μM of the STAT3 inhibitor S31-201 for 8 hours followed by 18 hour treatment with or without 10 μg/ml cetuximab. Mean +/− SD is shown for three independent experiments. * P < 0.05.

Article Snippet: Oxaliplatin was purchased from Hospira Ltd, irinotecan from Teva, cetuximab (Erbitux) from Merck, panitumumab (Vectibix) from Amgen, abemaciclib (LY2835219) from Abmole, STAT3 inhibitor (S3I-201) from Cayman chemicals, and thymidine from Sigma-Aldrich.

Techniques: Expressing, Western Blot, Staining