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Journal: PLOS Biology

Article Title: Dose-dependent activation of the Hippo pathway by Type I and Type III interferons suppresses tissue repair by human bronchial epithelial cells

doi: 10.1371/journal.pbio.3003615

Figure Lengend Snippet: (A) Model for relationship between LATS1/2, YAP, and P53 in inhibiting cell proliferation. (B) Subconfluent BCi were treated with IFNλ1 or IFNβ for 24 hours. Fold change in cell count from 0 to 24 hours is shown, with dashed line at 1 representing starting cell number. Graph shows mean and SEM of four replicates per condition, with means compared using Ordinary One-Way ANOVA test. (C) BCi were grown to confluence, followed by IFNλ1 or IFNβ pretreatment and wounding with a standardized linear scratch (600 µm). Fraction of wound area remaining was quantified over time to calculate AUC. Mean and SEM of four replicates per condition are shown, with AUCs compared using Ordinary One-Way ANOVA test. (D) siCtrl and siLATS1/2-transfected BCi were treated with IFNλ1 or IFNβ for 24 hours. Fold change in cell count from 0 to 24 hours is shown, with dashed line at 1 representing starting cell number. Graph shows mean and SEM of four replicates per condition, with means compared using Ordinary One-Way ANOVA test. (E–G) BCi were transduced with an empty vector (EV) or YAP5SA. (E) western blot on EV and YAP5SA BCi lysates for Flag-tag. (F) EV and YAP5SA BCi were grown to confluence, followed by IFNλ1 or IFNβ treatment and wounding with a standardized linear scratch (600 µm). Fraction of wound area remaining was quantified. Graph shows mean and SEM of three replicates per condition, with means compared using Ordinary One-Way ANOVA test. (G) Subconfluent EV and YAP5SA BCi were treated with IFNλ1 or IFNβ for 24 hours. Fold change in cell count from 0 to 24 hours is shown, with dashed line at 1 representing starting cell number. Graphs show mean and SEM of four replicates per condition, with means compared using Ordinary One-Way ANOVA test. (H) western blot for p53 on cell lysates of siCtrl and siTP53-transfected HBEC four days after transfection. (I) siCtrl and siTP53 BCi were treated with IFNλ1 or IFNβ for 24 hours. Fold change in cell count from 0 to 24 hours is shown, with dashed line at 1 representing starting cell number. Graphs show mean and SEM of three replicates per condition, with means compared using Ordinary One-Way ANOVA test. (J) siCtrl and siTP53 HBEC were grown to confluence, followed by IFNλ1 (red) or IFNβ (blue) treatment and wounding with a standardized linear scratch (600 µm). Fraction of wound area remaining was quantified over time to calculate AUC. Graph shows mean and SEM of four replicates per condition, with AUCs compared using Ordinary One-Way ANOVA test. The data underlying this figure can be found in and .

Article Snippet: Basolateral media was collected for LDH assay (Cayman Chemical #601170, as described in manufacturer’s protocol, with values calculated from standard curve of lysed cells), Type I IFN reporter assay (InvivoGen, Cat# hkb-ifnabv2), and Type III IFN ELISA (PBL Assay Science, Cat# 61840).

Techniques: Cell Characterization, Transfection, Transduction, Plasmid Preparation, Western Blot, FLAG-tag

Journal: PLOS Biology

Article Title: Dose-dependent activation of the Hippo pathway by Type I and Type III interferons suppresses tissue repair by human bronchial epithelial cells

doi: 10.1371/journal.pbio.3003615

Figure Lengend Snippet: (A) Type III IFN receptor signaling and targets of ruxolitinib (rux) and fludarabine (flud). (B) Subconfluent HBEC were pretreated with rux or flud prior to IFNλ1 treatment, followed by western blot on cell lysates pLATS1 Ser 909 and total LATS1. (C, D) Subconfluent HBEC were pretreated with rux or flud prior to treatment with IFNλ1 for 24 hours, followed by RNA isolation and RT-qPCR for CYR61 or BIRC5 . Graphs show mean and SEM of three replicates per condition, with means compared using Ordinary One-Way ANOVA test. (E) Confluent rux- and flud-pretreated HBEC were treated with IFNλ1 followed by wounding with a standardized linear scratch (600 µm). Fraction of wound area remaining was quantified over time to calculate AUC. Graph shows mean and SEM of four replicates per condition, with means compared using Ordinary One-Way ANOVA test. (F–L) Rux- or flud-pretreated HBEC treated with IFNλ1 were fixed four hours after wounding with a standardized linear scratch (600 µm) and stained for YAP and DAPI. (F) Number of cells with nuclear YAP within 100µm of the leading edge of the scratch were quantified for each condition. Graph shows mean and SEM of six high-power fields of view per condition, with means compared using Ordinary One-Way ANOVA test. (G–L) For low magnification images, scale bar = 100 µm. Solid white line represents scratch border. Insets represent field of view captured by dashed white box in low magnification images and show merged channel (bottom) or individual YAP (red) and DAPI (blue) channels. For insets, scale bar = 20 µm. (M–O) Subconfluent siCtrl or siJAK1 HBEC were treated with IFNλ1 (M, N) for 24 hours, followed by RNA isolation and RT-qPCR for YAP target genes, or (O) for six hours, followed by western blot on cell lysates for pLATS1 Ser 909 and total LATS1. (P) siCtrl or siJAK1 HBEC were grown to confluence, followed by IFNλ1 pretreatment and wounding with a standardized linear scratch (600 µm). Fraction of wound area remaining was quantified over time to calculate AUC. Graphs show mean and SEM of four replicates per condition, with means compared using Ordinary One-Way ANOVA test. The data underlying this figure can be found in , , and at https://doi.org/10.17632/6rvkfsy2n8.1 .

Article Snippet: Basolateral media was collected for LDH assay (Cayman Chemical #601170, as described in manufacturer’s protocol, with values calculated from standard curve of lysed cells), Type I IFN reporter assay (InvivoGen, Cat# hkb-ifnabv2), and Type III IFN ELISA (PBL Assay Science, Cat# 61840).

Techniques: Western Blot, Isolation, Quantitative RT-PCR, Staining

Journal: PLOS Biology

Article Title: Dose-dependent activation of the Hippo pathway by Type I and Type III interferons suppresses tissue repair by human bronchial epithelial cells

doi: 10.1371/journal.pbio.3003615

Figure Lengend Snippet: (A) Subconfluent HBEC were treated with IFNλ1 (red) or IFNβ (blue) for 6 hours at specified concentrations, followed by western blot on cell lysates for pLATS1 Ser 909 , total LATS1, pSTAT1 Tyr 701 , and total STAT1. Top three panels show western blot for pLATS1/LATS1/β actin and bottom three panels show separate western blot on same cell lysates for pSTAT1/STAT1/β actin. (B) Confluent HBEC were treated with IFNλ1 (red) or IFNβ (blue) followed by wounding with a standardized linear scratch (600µm). Fraction of wound area remaining was quantified over time to calculate AUC. Graph shows mean and SEM of three replicates per condition as a function of IFN concentration. AUCs at each IFN concentration were compared using Ordinary One-Way ANOVA test, with red and blue p-values representing comparison between no IFN and IFNλ1 or IFNβ treatment, respectively. (C–G) Subconfluent HBEC were treated with IFNλ1 or IFNλ2 at specified concentrations for (C) 6 or (D–G) 24 hours, followed by (C) western blot on cell lysates for pLATS1 Ser 909 , total LATS1, pSTAT1 Tyr 701 , and total STAT1, or (D–G) RNA isolation and RT-qPCR for ISGs and YAP target genes. Graphs show mean and SEM of four replicates per condition, with means compared using (D, E) Ordinary One-Way ANOVA or (F, G) Brown–Forsythe and Welch ANOVA tests. (H–N) Confluent HBEC were treated with IFNλ2 or IFNλ1 at specified concentrations prior to (H, J) wounding with a standardized linear scratch (600 µm) or (I, K–N) infection with RV or IAV. Photomicrographs show (H) remaining wound area at 0 and 24 hours after scratch and (I) cytopathic effects of viral infection with or without IFNλ2 pretreatment. For all images, scale bar = 100 µm. (J) Fraction of wound area remaining was quantified over time to calculate AUC. (K–N) RV and IAV replication by RT-qPCR and plaque assay at 72 hours post infection. Graphs show mean and SEM of four replicates per condition and were compared by (J) Ordinary One-Way ANOVA test or (K–N) Welch’s t test. The data underlying this figure can be found in and .

Article Snippet: Basolateral media was collected for LDH assay (Cayman Chemical #601170, as described in manufacturer’s protocol, with values calculated from standard curve of lysed cells), Type I IFN reporter assay (InvivoGen, Cat# hkb-ifnabv2), and Type III IFN ELISA (PBL Assay Science, Cat# 61840).

Techniques: Western Blot, Concentration Assay, Comparison, Isolation, Quantitative RT-PCR, Infection, Plaque Assay

Journal: PLOS Biology

Article Title: Dose-dependent activation of the Hippo pathway by Type I and Type III interferons suppresses tissue repair by human bronchial epithelial cells

doi: 10.1371/journal.pbio.3003615

Figure Lengend Snippet: (A) Conditions that signal cell confluence lead to activation and phosphorylation of MST1/2. Active MST1/2 phosphorylate and activate LATS1/2, which then phosphorylate and inactivate YAP, reducing cell proliferation and migration. (B) Conditions that signal subconfluence to cells, such as a wound caused by an injury, results in increased YAP nuclear activity, promoting cell proliferation and migration. (C) During acute viral infection, high concentrations of Type III and/or Type I IFN receptor signaling trigger JAK-dependent, STAT1-independent phosphorylation of LATS1, leading to phosphorylation and degradation of YAP and reduced tissue repair activities mediated by YAP target genes. In rapidly proliferating cell types, active LATS1/2 also blocks repair in part via a p53-dependent mechanism. (D) During resolution of viral infection, lower concentrations of Type III and/or Type I IFN may continue to activate STAT1-mediated antiviral defenses but no longer activate LATS1/2, allowing epithelial repair to resume. (E–F) Time course of expected LATS-1 and STAT-1 phosphorylation for viral infections with different kinetics. Dotted lines indicate threshold IFN concentration required for LATS1 activation (high threshold) or STAT1 activation (lower threshold). (E) LATS1-P and STAT1-P time course expected for acute viral infection in which viral load and IFN induction peak at day 1 and then decline. (F) LATS1-P and STAT1-P time course expected for viral infection that continues to increase in viral load and IFN induction between days 1 and 5 post-infection. Made with Biorender.com.

Article Snippet: Basolateral media was collected for LDH assay (Cayman Chemical #601170, as described in manufacturer’s protocol, with values calculated from standard curve of lysed cells), Type I IFN reporter assay (InvivoGen, Cat# hkb-ifnabv2), and Type III IFN ELISA (PBL Assay Science, Cat# 61840).

Techniques: Activation Assay, Phospho-proteomics, Migration, Activity Assay, Infection, Concentration Assay