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: After treatment, cells were fixed with 4% paraformaldehyde (10min), permeablized with 0.1% Triton X-100 (10min), AJP-Lung Cell Mol Physiol doi:10.1152/ajplung.00127.2021 www.ajplung.org L577 Downloaded from journals.physiology.org/journal/ajplung (2405:4803:DB54:6CA0:48BC:7FD6:D8FE:BCC7) on March 11, 2025. blocked with 5% goat serum in PBS (2h), and incubated with primary antibody in blocking buffer overnight at 4 C. Primary antibodies used were anti-ATF6 (Cat. No. 24169-1-AP at VWR, 1:50 dilution), SNAI1 (Cat. No. 3895S at Cell Signaling, 1:50 dilution), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) (Abcam, Cat. No. ab190966 at Abcam, 1:100 dilution), VIM (Cell Signaling, Cat. No. 5741S, 1:100 dilution), IRE1a (Abcam Cat. No. ab37073, 1:50 dilution), Calnexin (Abcam Cat. No. ab22595, 1:1,000 dilution), and GOLG2a/GM130 (Abcam Cat. No. ab52649, 1:200 dilution).

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: After treatment, cells were fixed with 4% paraformaldehyde (10min), permeablized with 0.1% Triton X-100 (10min), AJP-Lung Cell Mol Physiol doi:10.1152/ajplung.00127.2021 www.ajplung.org L577 Downloaded from journals.physiology.org/journal/ajplung (2405:4803:DB54:6CA0:48BC:7FD6:D8FE:BCC7) on March 11, 2025. blocked with 5% goat serum in PBS (2h), and incubated with primary antibody in blocking buffer overnight at 4 C. Primary antibodies used were anti-ATF6 (Cat. No. 24169-1-AP at VWR, 1:50 dilution), SNAI1 (Cat. No. 3895S at Cell Signaling, 1:50 dilution), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) (Abcam, Cat. No. ab190966 at Abcam, 1:100 dilution), VIM (Cell Signaling, Cat. No. 5741S, 1:100 dilution), IRE1a (Abcam Cat. No. ab37073, 1:50 dilution), Calnexin (Abcam Cat. No. ab22595, 1:1,000 dilution), and GOLG2a/GM130 (Abcam Cat. No. ab52649, 1:200 dilution).

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

Journal: Science advances

Article Title: A gut-brain axis mediates sodium appetite via gastrointestinal peptide regulation on a medulla-hypothalamic circuit.

doi: 10.1126/sciadv.add5330

Figure Lengend Snippet: Fig. 2. Colon mesenchymal senses hyponatremia by biosynthesis of SCT. (A) Confocal images showing SCT expression in the colon, small intestine, and duodenum using SCT-Cre;ROSA-tdTomato double-transgenic mice. Scale bars, 500 μm. (B) Colon mRNA level of SCT was increased under Na-D conditions. Two-way ANOVA, group factor F3,32 = 32.57, P < 0.0001. (C) Na-D–induced colon secretion of SCT. F2,25 = 388.6, n = 5 mice for each group in (B) and (C). (D and E) Confocal images showing colon SCT+ within enterochromaffin (TPH+) cells, but not myofibroblasts/fibroblasts (vimentin+). Scale bars, 200 μm (left) and 50 μm (right). DAPI, 4′,6-diamidino-2-phenylindole. (F) Schematic diagram of colon-specific KD of SCT. (G) Specific infection of siRNA-SCT-GFP virus in colon tissue. Scale bars, 200 μm. (H) mRNA level of SCT in the colon, small intestine, and duodenum under Na-S, Na-D, SCT KD in colon + normal diet (KD + Na-S), or KD + Na-D. F2,50 = 1.343, P < 0.05. (I) Na-D–induced SCT release was blocked by colon-specific SCT KD. F3,48 = 2.221, P < 0.05. (J) Na-D increased serum SCT, which was absent under colon-specific SCT KD. One-way ANOVA, F3,5.295 = 31.62, P < 0.001. n = 5 mice for each group in (H) to (J). (K) The Na-D-induced CSF SCT surge was not present in the colon-specific SCT KD group. n = 5 mice for each group. (L) Comparison of 1-hour cumulative saline intake. (M) Quantification of water and 3% saline intake during the 1-hour two-bottle test. SCT KD in colons led to decreased 3% saline intake. n = 5 mice for each group in (L) and (M). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, with significant difference. All data were presented as means ± SD.

Article Snippet: Sections were then incubated for 24 hours with primary antibodies diluted in blocking buffer with 0.3% Triton X-100: sheep anti-TPH (1:500, Millipore, AB1541), vimentin XP rabbit monoclonal antibody (Alexa Fluor 48 conjugate) (1:400, Cell Signaling, 9854), guinea pig anti–c-Fos (1:500, Synaptic Systems, 226005), rabbit anti-MC4R (1:1000, Abcam, ab24233), rabbit anti-oxytocin (1:10,000, Immunostar, AB_572258), rabbit anti-vasopressin (1:1000, Millipore, AB1565), rabbit antiHSD11B2 (1:500, Proteintech, 14192-1-AP), rabbit anti-mcherry antibody (DsRed) (1:500, Clontech, Mountain View, CA), and anti-GFP antibody (1:500, Aves Labs, GFP-1020).

Techniques: Expressing, Transgenic Assay, Infection, Virus, Comparison, Saline

Journal: International Journal of Molecular Medicine

Article Title: Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells

doi: 10.3892/ijmm.2015.2222

Figure Lengend Snippet: Inhibition of hyaluronan (HA) by 4-methylumbelliferone (4-MU) abolishes the transforming growth factor-β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT). (A and B) Western blot analysis of the expression of downstream pathways and EMT-related proteins, such as E-cadherin, vimentin, Snail and Twist, following stimulation with TGF-β1 or TGF-β1 plus 4-MU. β-actin was used as a loading control. (C) A Transwell assay was carried out to determine the migratory/invasive ability of the cells following stimulation with TGF-β1 and treatment with 4-MU. All graphs represent the means ± SD of 3 independent experiments. The y-axis represents the fold change in the number of cells. * P<0.05 vs. control.

Article Snippet: The following primary antibodies were used: E-cadherin (#3195s), ZEB-1 (#6935s), N-cadherin (#4061s), Snail (#5879s), vimentin (#7391s) (all from Cell Signaling Technology, Beverly, MA, USA), Twist (ab50581; Abcam, Cambridge, UK), EGFR (#4267s), phosphorylated (p-)AKT (#4060s), p-extracellular signal-regulated kinase (ERK; #4370s), p-Smad (#3101s), ERK (#4695), Smad (#5339s) (all from Cell Signaling Technology), β-actin (Sigma-Aldrich), AKT (#2920s; Cell Signaling Technology) and CD44 (sc-18849; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Inhibition, Western Blot, Expressing, Control, Transwell Assay

Journal: International Journal of Molecular Medicine

Article Title: Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells

doi: 10.3892/ijmm.2015.2222

Figure Lengend Snippet: shRNA targeting CD44 (shCD44) blocks the activation of the AKT and ERK pathways and causes the reversal of epithelial-mesenchymal transition (EMT). (A) Western blot analysis revealed that the CD44 protein levels and epidermal growth factor receptor (EGFR) expression were altered following stimulation with transforming growth factor-β1 (TGF-β1) or TGF-β1 plus shRNA. β-actin was used as a loading control. (B and C) Western blot analysis of the expression of downstream pathways and EMT-related proteins, such as E-cadherin, vimentin, Snail and Twist, following stimulation with TGF-β1 or TGF-β1 plus shCD44. β-actin was used as a loading control.

Article Snippet: The following primary antibodies were used: E-cadherin (#3195s), ZEB-1 (#6935s), N-cadherin (#4061s), Snail (#5879s), vimentin (#7391s) (all from Cell Signaling Technology, Beverly, MA, USA), Twist (ab50581; Abcam, Cambridge, UK), EGFR (#4267s), phosphorylated (p-)AKT (#4060s), p-extracellular signal-regulated kinase (ERK; #4370s), p-Smad (#3101s), ERK (#4695), Smad (#5339s) (all from Cell Signaling Technology), β-actin (Sigma-Aldrich), AKT (#2920s; Cell Signaling Technology) and CD44 (sc-18849; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: shRNA, Activation Assay, Western Blot, Expressing, Control