Journal: PLoS ONE

Article Title: The Effects of Knockdown of Rho-Associated Kinase 1 and Zipper-Interacting Protein Kinase on Gene Expression and Function in Cultured Human Arterial Smooth Muscle Cells

doi: 10.1371/journal.pone.0116969

Figure Lengend Snippet: (A) CASMCs were transfected with siRNA to ZIPK or ROCK1 or with negative control siRNA. Cells were lysed 48 h later for qRT-PCR to quantify IL-6 and IL-1β mRNA levels. Values represent means ± SEM ( n = 7 for IL-6 and n = 6 for IL-1β). *significantly different from control (p < 0.001 except for IL-1β in ZIPK knockdown where p = 0.007). (B) IL-6 levels in the medium of control cells and cells transfected with ROCK1 or ZIPK siRNA were quantified by ELISA. Values represent means ± SEM ( n = 20 for CASMC, n = 29 for UASMC). *significantly different from control ( p < 0.002).

Article Snippet: IL-6 levels were quantified in culture supernatants of CASMC and UASMC 48 h after siRNA transfection using an ELISA kit (Human IL-6 DuoSet, DY206, R&D Systems) according to the manufacturer’s instructions.

Techniques: Transfection, Negative Control, Quantitative RT-PCR, Control, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: siRNA Library Screening Identifies a Druggable Immune-Signature Driving Esophageal Adenocarcinoma Cell Growth

doi: 10.1016/j.jcmgh.2018.01.012

Figure Lengend Snippet: LIF hierarchically regulates STAT3 phosphorylation, IL6 expression, and cell proliferation in EAC cells. SiRNA-mediated silencing of LIF expression results in reduced LIF mRNA ( A ) expression, ( B ) secretion, and ( C ) a recombinant-recoverable reduction in cell proliferation in CP-D, SKGT4, and OE-33 EAC cell lines. Treatment of LIF silenced cells with recombinant LIF protein (rLIF) results in ( D ) recovery of LIF mRNA in CP-D, SKGT4, and OE33 EAC cells and ( E ) protein expression levels in SKGT4 EAC cells. ( F ) STAT3-Y705 phosphorylation levels in LIF-silenced SKGT4 EAC cells are rescued by treatment with recombinant LIF protein as determined by Western blot. ( G ) Silencing LIF expression in SKGT4 EAC cells results in decreased IL6 mRNA expression that is recovered upon exposure to rLIF protein. ( H ) Neither silencing IL6 expression by siRNA-mediated silencing nor treatment with anti-IL6 antibody affects LIF mRNA levels or SKGT4 EAC cell growth. ( I ) Silencing LIF expression results in decreased expression of C1QA mRNA but not TREM2 in SKGT4 EAC cells. ∗ P < .05, ** P < .01, *** P < .001, and **** P < .0001 in either Student t test (proliferation) or Mann–Whitney testing (mRNA expression). siNT, nontargeting siRNA pool; siLIF, LIF-targeted siRNA pool; rLIF, recombinant LIF protein; rIL6, recombinant IL6 protein.

Article Snippet: Treatments with recombinant leukemia inhibitory factor (LIF) (30 ng/mL, ab57665; Abcam), IL6 (30 ng/mL; R&D Systems, Minneapolis, MN), native C1q (75 μg/mL; Sigma, Oakville, Ontario), and IL6 blocking antibody (AB206NA) experiments were performed in either 6- or 96-well cell culture plates for protein and viability experiments, respectively.

Techniques: Phospho-proteomics, Expressing, Recombinant, Western Blot, MANN-WHITNEY