calu3 cells  (ATCC)

Journal: Anesthesiology

Article Title: Hypoxia-inducible Factor 1α–dependent Induction of Alveolar miR-147b and Direct Targeting of Viral RNA in SARS-CoV-2–associated Acute Respiratory Distress Syndrome

doi: 10.1097/ALN.0000000000006306

Figure Lengend Snippet: HIF1A-dependent induction of miR-147b during alveolar injury and SARS-CoV-2 infection. ( A ) Expression level of hsa-miR-147b in human primary alveolar epithelial cells after cyclic mechanical stretch (30%). n = 3. Mean ± SD is shown. ( B ) Transcription factor binding array of miR-147b promoter in Calu-3 cells 2 h after cyclic mechanical stretch (30%). ( C ) Schematic of promoter construct of miR-147 demonstrating HRE and NF-κB responsive elements. ( D ) Plasmid with the constructs shown in c was transfected into Calu-3 cells for 24 h. Dual-luciferase activity was measured after 18 h of mechanical stretch (Firefly/ Renilla ). n = 3. Mean ± SD is shown. ( E ) Chromatin immunoprecipitation (ChIP) assay to examine for direct binding of HIF1A to the HRE regions within the hsa-miR-147b promoter regions during mechanical stretch (8 h) in Calu-3 cells (gels represent two individual experiments). ( F ) Calu-3 cells were exposed to ambient hypoxia (1% oxygen) and expression level of hsa-miR-147b was measured by RT-qPCR. n = 6. Mean ± SD is shown. * P < 0.05. One-way ANOVA. ( G ) A549 cells were exposed to ambient hypoxia (1% oxygen), and expression level of hsa-miR-147b was measured by RT-qPCR. n = 5. Mean ± SD is shown. * P < 0.05; ** P < 0.01. One-way ANOVA. ( H ) Calu-3 cells were treated with dimethyloxaloglycine (DMOG; 1 µm, 24 h), and expression level of hsa-miR-147b was measured by RT-qPCR. n = 6. Mean ± SD is shown. ** P < 0.01. Mann–Whitney test. ( I ) A549 cells were treated with DMOG (1 µm, 24 h), and expression level of hsa-miR-147b was measured by RT-qPCR. n = 5. Mean ± SD is shown. ** P < 0.01. Mann–Whitney test. ( J ) Protein levels of HIF1A or HIF2A in A549-hACE2 cells after SARS-CoV-2 infection (0.1 MOI) by Western blot. ( K and L ) Quantification of HIF1A and HIF2A in A54-hACE2 cells after SARS-CoV-2 infection (0.1 MOI). n = 9. Mean ± SD is shown. ns P > 0.05; ** P < 0.01; **** P < 0.0001. One-way ANOVA. ( M ) A549-hACE2 cells with siRNA-mediated repression of HIF1A were exposed to SARS-CoV-2 infection (WA1; 0.2 MOI) or mock-infected. Hsa-miR-147b transcript levels were examined by real-time RT-PCR. n = 3 or 4. Mean ± SD is shown. hACE2,human angiotensin-converting enzyme 2; HIF, hypoxia-inducible factor; HRE, hypoxia-response element; MOI, multiplicity of infection; NF, nuclear factor; ns, not significant; RT-PCR, reverse-transcription polymerase chain reaction; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; siRNA, small interfering RNA; WA1, Washington-1.

Article Snippet: Calu-3 human airway epithelial cells, HEK-293 cells or human pulmonary epithelial A549 cells were purchased from American Type Culture Collection (USA; HTB-55, RRID:CVCL_0609; CRL-1573; CCL-185).

Techniques: Infection, Expressing, Binding Assay, Construct, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Small Interfering RNA