Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

doi: 10.1152/ajplung.00127.2021

Figure Lengend Snippet: Figure 4. IRE1a-XBP1 pathway regulates HBP and EMT gene networks: IRE1a kinase inhibitor. hSAECs were infected with RSV (MOI = 1.0) for 24h in the absence (DMSO, solvent carrier) or presence of the IRE1a endoribonuclease inhibitor KIRA8 (KIRA) or the ATF6 inhibitor ceapin-A7 (A7) at 10mM. Shown is fold change of mRNA relative to mock infection determined by Q-RT-PCR. Bars represent means ± ranges plus all data points of three independent experiments. GFPT2 (A); IL6 (B); SNAI1, ZEB1, VIM, and FN1 (C); MMP9 (D); and effect on RSV transcription (E). Note the equivalent expression of RSV N transcript between treatments indicates that RSV replication was not significantly affected by either KIRA8 or ceapin-A7. F: effect on RSV infectivity. Shown are focus forming units (FFU) determined by colorimetric assay using polyclonal anti-RSV antibodies. P < 0.05, P < 0.01, post hoc Tukey’s pairwise comparison. EMT, epithelial mesenchymal transition; HBP, hexosamine biosynthetic pathway; hSAECs, human small airway epithelial cells; IRE1a-XBP1, inositol-requiring enzyme 1a-X-box binding protein 1; Q-RT-PCR, quantitative reverse transcription polymerase chain reaction; RSV, respira- tory syncytial virus; ZEB1, zinc finger E-box binding homeobox 1.

Article Snippet: The slides were blocked in 10% goat serum plus mouse onmouse Ig blocking reagent (MOM, Vector, Canada) in Tris-buffered saline (TBS) overnight at 4 C and incubated with SNAI1 antibody (Cat. No. 3895S at Cell Signaling, 1:100 dilution) or control IgG at 4 C overnight afterwards.

Techniques: Infection, Solvent, Reverse Transcription Polymerase Chain Reaction, Expressing, Colorimetric Assay, Comparison, Binding Assay, Reverse Transcription, Polymerase Chain Reaction, Virus

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

doi: 10.1152/ajplung.00127.2021

Figure Lengend Snippet: Figure 5. IRE1a-XBP1 pathway regulates HBP and EMT gene networks: shRNA silencing. Q-RT-PCR analysis of hSAECs stably expressing nontargeting shRNA (Luc), IRE1a-targeting shRNA (IRE1), or XBP1-targeting shRNA (XBP1). Cells were mock or RSV-infected (MOI= 1, 24 h). Shown is fold change of mRNA relative to mock infection determined by Q-RT-PCR. Bars represent means ± ranges plus all data points of three independent experiments. P < 0.01. A: XBP1-Total; B: IRE1a; C: XBP1s; D: GFPT2; E: SNAI1; F: IL6; G: FN1; H: VIM; I: MMP9; J: RSV N transcription; K: RSV infectivity by colorimetric mea- surement of FFUs. EMT, epithelial mesenchymal transition; HBP, hexosamine biosynthetic pathway; hSAECs, human small airway epithelial cells; IRE1a inositol-requiring enzyme 1a; MOI, multiplicity of infection; Q-RT-PCR, quantitative reverse transcription polymerase chain reaction; RSV, respiratory syn- cytial virus; SNAI1, Snail family transcriptional repressor 1; XBP1, X-box binding protein 1.

Article Snippet: The slides were blocked in 10% goat serum plus mouse onmouse Ig blocking reagent (MOM, Vector, Canada) in Tris-buffered saline (TBS) overnight at 4 C and incubated with SNAI1 antibody (Cat. No. 3895S at Cell Signaling, 1:100 dilution) or control IgG at 4 C overnight afterwards.

Techniques: shRNA, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Expressing, Infection, Reverse Transcription, Polymerase Chain Reaction, Virus, Binding Assay

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

doi: 10.1152/ajplung.00127.2021

Figure Lengend Snippet: Figure 6. XBP1 is a direct regulator of IL6, SNAI1, and GFPT2 genes. A, E, I, and M are integrated genomics viewer (IGV) of the individual ATAC-seq peaks for uninfected (Mock) and RSV-infected hSAECs. Trans- posase peaks are indicated by the height of the red graphs and located relative to each gene. Note the dramatic increase in transposase cleavage in the regulatory regions in response to RSV. For the XChIP assays in this study, hSAECs were infected with RSV (MOI=1.0) for 24 h in the absence or presence of the IRE1a RNase inhibitor KIRA8 at 10μM (KIRA). B, F, J, and N are standard curves for the performance of the quantitative genomic PCR (Q-gPCR) ampli- fying the genomic DNA regions of IL6 pro- moter (B), SNAI1 promoter (F), SNAI1 30UTR (J), and GFPT2 intragenic superenhancer (N) (see the PCR primers in Table 2). Increasing amounts of genomic DNA iso- lated from hSAECs were applied. Note that each curve has a regression line with R2 > 0.92. C, G, K, and O are XChIP assays for XBP1 binding to indicated genes. The genomic DNA enrichment by XBP1 XChIP was assayed by Q-gPCR using primers specific for IL6 promoter (C), SNAI1 pro- moter (G), SNAI1 30UTR (K), and GFPT2 intragenic superenhancer (O). Data are cal- culated as fold change relative to unin- fected hSAECs (DMSO) and plotted as means ± ranges plus all data points of duplicate independent immunoprecipi- tates. D, H, L, and P are XChIP assays of RNA polymerase II (Pol II) binding to indi- cated genes. Q-gPCR assays were per- formed as in C, G, K, and O. IgG controls of the XChIP had less than 1/10 Q-gPCR signal relative to XChIP with either XBP1 or RNA Pol II antibodies (not shown). P < 0.01, ANOVA with post hoc Tukey’s comparison. ATAC-seq, assay for transposase-accessi- ble chromatin using sequencing; hSAECs, human small airway epithelial cells; MOI, multiplicity of infection; RSV, respiratory syncytial virus; SNAI1, Snail family transcrip- tional repressor 1; XBP1, X-box binding pro- tein 1.

Article Snippet: The slides were blocked in 10% goat serum plus mouse onmouse Ig blocking reagent (MOM, Vector, Canada) in Tris-buffered saline (TBS) overnight at 4 C and incubated with SNAI1 antibody (Cat. No. 3895S at Cell Signaling, 1:100 dilution) or control IgG at 4 C overnight afterwards.

Techniques: Infection, Binding Assay, Comparison, Sequencing, Virus