control plasmid encoding gpf shrna  (OriGene)

Journal: Carcinogenesis

Article Title: Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation

doi: 10.1093/carcin/bgr075

Figure Lengend Snippet: MSLN overexpression leads to increased IL-6 production in PC cells. (A). MIA, MIA-V, MIA-GFP and MIA-MSLN cells were seeded at 1 × 106 cells per well in six-well plates and cultured until 60% confluence. Thereafter, the medium was replaced and the supernatants were harvested at 48 h of further incubation and the levels of IL-6 were determined by using the Luminex-based IL-6 assay kit. Y-axis represents IL-6 concentration in pg/ml. (B) Silencing MSLN using MSLN-specific shRNA plasmid. MIA-V and MIA-MSLN cells were transfected with MSLN-specific shRNA plasmids or GFP shRNA control plasmids. The cells collected 48 h posttransfection were used to detect MSLN mRNA by real-time polymerase chain reaction. The MSLN-expressing levels were detected by using real-time polymerase chain reaction. Y-axis represents MSLN mRNA level normalized to the GAPDH mRNA level. Relative mRNA level is presented as 2 [Ct(GAPDH)−Ct(IL-6)]. The bars denote SD of duplicate data. Experiment was performed several times using separate MIA-MSLN pools and different siRNA/shRNAs against MSLN with similar results. (C) Silencing MSLN decreases IL-6 mRNA production in MIA-MSLN cells. MIA-V and MIA-MSLN cells were transfected with MSLN-specific shRNA plasmids or GFP shRNA control plasmids. The cells collected 48 h posttransfection were used to detect IL-6 mRNA by real-time polymerase chain reaction. Y-axis represents IL-6 mRNA level relative to GAPDH. The bars denote SD of duplicate data. Experiment was performed several times using separate MIA-MSLN pools and different siRNA/shRNAs against MSLN with similar results. (D) Silencing MSLN decreases IL-6 secretion in MIA-MSLN cells. MIA-V and MIA-MSLN cells were transfected with MSLN-specific siRNA or scrambled siRNA. The cells collected 48 h posttransfection from 24-well plates in 2 ml of medium were used to detect IL-6 by Luminex-based IL-6 assay kit. Values on Y-axis show the amount of IL-6 in pg/ml, bars denoting SD of duplicate data. (E) Stable overexpression of MSLN in human PC cell Panc1. The GAPDH-normalized MSLN expression levels in the stably MSLN expressing (Panc1-MSLN) and control MIA cells generated by retroviral gene transfer and subsequent puromycin selection are shown. (F) Panc1, Panc1-V, Panc1-GFP and Panc1-MSLN cells were seeded at 1 × 106 cells per well in six-well plates and cultured until 80% confluence, cells collected were used to detect IL-6 mRNA by real-time polymerase chain reaction. Y-axis represents IL-6 mRNA level relative to GAPDH. The bars denote SD of duplicate data. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test. * and # denote comparison to different base lines and hence are denoted differently.

Article Snippet: For MSLN shRNA transfection experiments, a plasmid encoding MSLN shRNA (TR311377; Origene, Rockville, MD) and a control plasmid encoding GPF shRNA (TR30003) were used.

Techniques: Over Expression, Cell Culture, Incubation, Luminex, Concentration Assay, shRNA, Plasmid Preparation, Transfection, Control, Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Generated, Retroviral, Selection, Comparison

Journal: Carcinogenesis

Article Title: Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation

doi: 10.1093/carcin/bgr075

Figure Lengend Snippet: IL-6 is an autocrine growth and survival factor for the MIA-MSLN cells. (A) Overexpression of MSLN promotes PC cell proliferation in serum-free conditions. MIA-MSLN and control MIA-V cells were seeded in 96-well plates (2 × 103 cells per well), serum starved (0% FBS) for 24 h before changing to fresh serum-free medium and cultured for 6 days. Viability was measured with MTT. Relative increase in viability was measured by dividing viability at a time point by viability of same cells at day 0 (day after plating) and is plotted along Y-axis. Data plotted show mean of triplicate wells. Proliferation after initial starvation followed by low serum supplementation was performed several times, serum-free survival was also performed multiple times, and data shown is a representative one. (B) Serum starvation increases IL-6 production by MIA-MSLN but not by MIA-V cells. MIA-V or MIA-MSLN cells (4 × 106) were plated in T75 flasks and cultured till 90% confluent. Growth medium was then replaced by serum-free medium and supernatants were collected after 24 and 48 h and assayed for IL-6 using the Luminex-based IL-6 assay kit. Values on Y-axis show the amount of IL-6 in pg/ml, bars denoting SD of duplicate data. (C) IL-6 siRNA reduced MIA-MSLN cell proliferation. MIA-V and MIA-MSLN cells were transfected in six-well plates using a pool of four IL-6-specific siRNAs or a negative control pool of scrambled siRNAs. At 24 h after the transfection, cells were trypsinized, counted and seeded in 96-well plates (2 × 103 cells per well), serum starved (0% FBS) for 24 h before changing to fresh medium with 0.2% FBS and cultured for 2 days. Viability was measured with MTT. Relative increase in viability was measured by dividing viability at a time point by viability of same cells at day 0 (day after plating) and is plotted along Y-axis. Data plotted show mean of triplicate wells. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test. Experiment was performed several times using various serum conditions with similar results shown is a representative data. (D) IL-6 siRNA reduced cell cycle distribution of MIA-MSLN cells. MIA-MSLN cells treated with IL-6-specific siRNA as previously were allowed to grow in serum-free media for 24 h after initial growth media replenishing for 12 h posttransfection and collected for Propidium Iodide (PI) staining. The numbers of cells in each phase are plotted along the Y-axis. (E) IL-6 siRNA transfected and control MIA-MSLN cells were continued in culture for 96 h and subjected to Caspase3 activation detection by western blot. (F) In order to see which IL-6/stat3 regulated genes were affected by IL-6 siRNA treatment, control and siIL-6 treated MIA-MSLN cells were subject to western blot detection of various IL-6/stat3 regulated genes.

Article Snippet: For MSLN shRNA transfection experiments, a plasmid encoding MSLN shRNA (TR311377; Origene, Rockville, MD) and a control plasmid encoding GPF shRNA (TR30003) were used.

Techniques: Over Expression, Control, Cell Culture, Luminex, Transfection, Negative Control, Staining, Activation Assay, Western Blot

Journal: Carcinogenesis

Article Title: Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation

doi: 10.1093/carcin/bgr075

Figure Lengend Snippet: MSLN/IL-6 axis provides a major survival signal in PC cells affecting growth under both anchorage-dependent and independent conditions. (A) Relative MSLN and IL-6 mRNA expression in selected PC cells. Total mRNA from the cell lines were reverse transcribed and tested for MSLN/IL-6 expression. The left hand side Y-axis denotes MSLN mRNA levels in each cell line normalized to the GAPDH mRNA level. Relative mRNA level is presented as 2 [Ct(GAPDH)−Ct(MSLN)]. The bars denote SD of duplicate data. Line graph represents the IL-6 mRNA in the same cell lines and the right hand side Y-axis indicates normalized IL-6 levels. (B) Effect of blocking MSLN on cell growth of multiple PC cells. Hs766T, BxPC3, both expressing MSLN/IL-6 and AsPC-1 with high MSLN and low IL-6 were treated with pools of MSLN-specific siRNA or non-targeting scrambled siRNA and plated as per reverse transcription protocol in 96-well plates in triplicates. After 36 h, complete media was replaced by serum-free media and then after another 24 h by 0.2% serum containing media and cultured for 5 days. Relative viability was measured by dividing viability of treated cells (treated with MSLN/scrambled siRNA) by that of mock-treated cells using MTT and is plotted along Y-axis. Data plotted show mean of triplicate wells. *, denote P < 0.05, and **, denote P < 0.01, compared with controls, t-test. (C) Stable MSLN-silenced AsPC-shMSLN cells and proliferation. Cells were seeded in 96-well plates (2 × 103 cells per well), serum starved (0% FBS) for 24 h before changing to fresh 0.2% FBS medium and cultured for 6 days. Viability was measured with MTT. Relative increase in viability was measured by dividing viability at a time point by viability of same cells at day 0 (day after plating) and is plotted along Y-axis. Data plotted show mean of triplicate wells. (D) Ability of MSLN-expressing cells to grow under anchorage-independent conditions. Cells were plated in ultra-low attachment plates and allowed to grow spheroid cultures for 8 days and then photographed. Shown are representative wells for each cell. (E) At 36 h posttransfection, BxPC3 cells treated with pools of MSLN/IL-6-specific siRNA or non-targeting scrambled siRNA in six-well plates were trypsinized and plated at 6000 cells per well of ultra-low attachment 96-well plates and cultured for 5 days before being photographed. (i) Spheroid colonies formed by BxPC3 ± IL-6/MSLN/scrambled siRNAs. (ii) Viability was measured by MTT. Relative viability was measured by dividing viability of treated cells (treated with MSLN/IL-6/scrambled siRNA) by that of mock-treated cells and is plotted along Y-axis. Data plotted show mean of triplicate wells. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test. (F) Exogenous rIL-6 induces proliferation in MSLN-expressing PC cells. Cells were seeded in 96-well plates (3 × 103 cells per well), serum starved (0% FBS) for 24 h and treated with IL-6 (100 ng/ml) ± sIL-6R (50 μg/ml) in serum-free medium for 5 days. Relative viability was measured by dividing viability after any treatment by viability of untreated cells by MTT and is plotted along Y-axis. Data plotted show mean of triplicate wells. Effect of blocking IL-6 and MSLN on growth of MSLN/IL-6-expressing cells under anchorage-dependent (G) conditions. Cell lines were treated with pools of IL-6-specific siRNA or non-targeting scrambled siRNA and plated as per reverse transcription protocol in 96-well plates in triplicates. After 36 h, serum-free media were added and then after another 24 h using 0.2% serum-containing media and cultured for 5 days. Relative viability was measured by dividing viability of treated cells (treated with IL-6/scrambled siRNA) by that of mock-treated cells using MTT and is plotted along Y-axis. Data plotted show mean of triplicate wells. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test.

Article Snippet: For MSLN shRNA transfection experiments, a plasmid encoding MSLN shRNA (TR311377; Origene, Rockville, MD) and a control plasmid encoding GPF shRNA (TR30003) were used.

Techniques: Expressing, Reverse Transcription, Blocking Assay, Cell Culture