Journal: Molecular and Cellular Pediatrics

Article Title: miRNA-221 is elevated in cystic fibrosis airway epithelial cells and regulates expression of ATF6

doi: 10.1186/s40348-014-0012-0

Figure Lengend Snippet: miR-221 is increased in trachea and bronchi and is predicted to target murine ATF6 3’UTR. (A) Relative expression of murine miR-145, miR-221 and miR-494 was determined by qRT-PCR using individual TaqMan murine miRNA assays and normalised to sno412 in the trachea and bronchi of 6-week-old wild-type ( n = 9) and βENaC-Tg ( n = 6) C57BL/6 mice. Data are represented as mean ± SEM and were compared by t test (* p < 0.01). All qRT-PCR experiments were performed in duplicate and included no-template controls. (B) Predicted binding locations of miR-221 in the full-length 5,422 bp murine ATF6 3′UTR and proposed base pair matches as predicted by TargetScan 6.2.

Article Snippet: HEK293 cells (1 × 10 5 in triplicate) were transiently co-transfected for 24 h with a WT-ATF6 3′UTR (OriGene, Rockville, MD, USA) firefly luciferase reporter vector containing the full-length 3′UTR (250 ng), a constitutive Renilla luciferase vector (100 ng) and 30 nM synthetic premiR mimics (PM) for miR-145, miR-221 and miR-494 (Applied Biosystems, Foster City, CA, USA) as indicated or with a scrambled control.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay

Journal: BMC Cancer

Article Title: Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells

doi: 10.1186/1471-2407-8-229

Figure Lengend Snippet: Effect of CS on proteolytic cleavage of ATF6 . Panel A: A549 cells were transfected with a plasmid expressing ATF6 (lanes 2 and 4, indicated by +), or a control plasmid (lanes 1 and 3), then treated with 2 mM DTT for 1 h (lanes 3 and 4) or left untreated (lanes 1 and 2). Western blots of whole cell lysates were probed with antibodies to ATF6 and α-tubulin. Panel B: All lanes show lysates from A549 cells transfected with a plasmid expressing ATF6. Cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to ATF6 and α-tubulin. Gel bands were quantified as described in the Methods section. CS treatment resulted in a 23% and 26% decrease in ATF6 90 kDa protein at 2 and 4 h respectively (lane 5 compared to lane 6, lane 7 compared to lane 8).

Article Snippet: At 24 hours after the seeding, cells were transfected with ATF6 expression plasmid ATF6/pCMV6-XL5 (OriGene Technologies, Inc., Rockville MD, Catalog # SC115551).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Endoplasmic reticulum stress regulates the innate immunity critical transcription factor interferon regulatory factor 3

doi: 10.4049/jimmunol.1102737

Figure Lengend Snippet: AEBSF, a site 1 protease inhibitor, blocks tunicamycin and 2-deoxyglucose induced IRF3 phosphorylation and tunicamycin-dependent synergistic IFN-β induction. A) RAW cells were pre-treated with 300 μM AEBSF for 1h, then stimulated with 1μM Tg for 2h, 20mM 2DG for 5h, or 10 μg/mL Tm for 5h. Cells were fixed and then stained with anti-pIRF3 (S386) plus secondary anti-rabbit Alexa Fluor488. Results are representative of 2 independent experiments. B) RAW cells were pretreated with AEBSF as in (A) and then untreated (NT), stimulated with Tg 1h, or Tm 5h, followed by an additional 3h media or LPS as indicated. Whole cell lysates were resolved by SDS PAGE and immunoblotted with anti-pIRF3 (S396), IRF-3 or actin. Results are representative of 2 independent experiments. C) RAW cells were pretreated with AEBSF as in (A), then stimulated with 1h Tg or 5h Tm followed by an additional 3h LPS as indicated. Relative IFN-β mRNA was quantified by qPCR with normalization to 18S rRNA. Results were combined from 4-5 independent experiments and error bars represent the standard error of the mean. * P<0.007. D) Cells were stimulated as in (B). Whole cell lysates were resolved by SDS PAGE and immunoblotted for precursor (P) and mature (M) cleaved forms of ATF6. Results are representative of 3 independent experiments.

Article Snippet: Antibodies β-actin, mouse mAb, NF-κB rabbit polyclonal Ab, and USF2 rabbit polyclonal Ab were from Santa Cruz; HA-Tag mouse mAb, IRF3 rabbit mAb (detects 1 IRF3 band) and phospho-IRF3 (pS396) rabbit mAb from Cell Signaling; Phospho-IRF3 (pS386) rabbit mAb from Epitomics; IRF3 rabbit polyclonal (detects 2 IRF3 forms), STING rabbit polyclonal Ab, and ATF6 mAb from Prosci; TBK1 rabbit mAb from Abcam; normal rabbit IgG from Millipore.

Techniques: Protease Inhibitor, Phospho-proteomics, Staining, SDS Page