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: Frontiers in pharmacology

Article Title: Autophagic Inhibition of Caveolin-1 by Compound Phyllanthus urinaria L. Activates Ubiquitination and Proteasome Degradation of β-catenin to Suppress Metastasis of Hepatitis B-Associated Hepatocellular Carcinoma.

doi: 10.3389/fphar.2021.659325

Figure Lengend Snippet: FIGURE 2 | CP inhibited migration and invasion of HBV-associated HCC. (A) HBV-associated cells including HepG2-HBx and HepG2-URG11 following CP administration and sorafenib treatment exhibited a significant delay in wound healing compared with that of control group. (B) CP treatment remarkably decreased the number of HBV-associated cells that migrated across matrigel and transwell chamber. (C) Western blot analysis showed that CP dramatically decreased expression levels of Vimentin and N-cadherin and increased E-cadherin level in both HepG2-HBx and HepG2-URG11 cells in a time-concentration dependent manner. All values represented the means ± SD (n 3, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Primary antibodies used in our study contained Cav1(16447-1-AP, Proteintech, Chicago, United States), N-cadherin (22018-1-AP, Proteintech, Chicago, United States), E-cadherin (20874-1-AP, Proteintech, Chicago, United States), Vimentin (10366-1-AP, Proteintech, Chicago, United States), URG11 (BS2170R, Beijing, China), β-catenin (66379-1-Ig, Proteintech, Chicago, United States), P-Akt (AF0016, Affinity, United States), Akt (60302-2-Ig, Proteintech, Chicago, United States), P-GSK3β (AB11002, AbSci, Baltimore, United States), GSK3β (BF0695, Affinity, United States) and β-actin (No.3700S, Cell Signaling Technology, CST, Boston, MA, United States).

Techniques: Migration, Control, Western Blot, Expressing, Concentration Assay

Journal: Frontiers in pharmacology

Article Title: Autophagic Inhibition of Caveolin-1 by Compound Phyllanthus urinaria L. Activates Ubiquitination and Proteasome Degradation of β-catenin to Suppress Metastasis of Hepatitis B-Associated Hepatocellular Carcinoma.

doi: 10.3389/fphar.2021.659325

Figure Lengend Snippet: FIGURE 8 | CP inhibited proliferation and migration of HBV-associated HCC ex vivo and in vivo. (A–C) HepG2-null and HepG2-HBx cells were grafted to the flank of nude mice by injecting the cells subcutaneously to observe tumor development, and volumes of tumors were determined at 3-day intervals. (D) Mice model with lung metastasis was established by tail vein injection of cancer cells. Upper panel, metastatic nodules in lungs. Lower panel, H&E staining of lung tissues. (E) Western blot analysis of tumor tissue showed that CP treatment decreased the expression levels of β-catenin and Cav-1, and enhanced the EMT process. (F) CP at concentrations from 60 to 120 μg/ml significantly decreased the primary as well as metastatic lesions in the zebrafish body when compared with control group. (G) H&E staining showed that CP suppressed the tumor growth in zebrafish. (H) Immunohistochemical staining of tumor in zebrafish showed CP treatment dramatically downregulated the expression levels of β-catenin, Cav-1 and Vimentin, and upregulated the expression level of E-cadherin. All values represented the means ± SD (n 6, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Primary antibodies used in our study contained Cav1(16447-1-AP, Proteintech, Chicago, United States), N-cadherin (22018-1-AP, Proteintech, Chicago, United States), E-cadherin (20874-1-AP, Proteintech, Chicago, United States), Vimentin (10366-1-AP, Proteintech, Chicago, United States), URG11 (BS2170R, Beijing, China), β-catenin (66379-1-Ig, Proteintech, Chicago, United States), P-Akt (AF0016, Affinity, United States), Akt (60302-2-Ig, Proteintech, Chicago, United States), P-GSK3β (AB11002, AbSci, Baltimore, United States), GSK3β (BF0695, Affinity, United States) and β-actin (No.3700S, Cell Signaling Technology, CST, Boston, MA, United States).

Techniques: Migration, Ex Vivo, In Vivo, Injection, Staining, Western Blot, Expressing, Control, Immunohistochemical staining

Journal: British journal of pharmacology

Article Title: A novel compound, denosomin, ameliorates spinal cord injury via axonal growth associated with astrocyte-secreted vimentin.

doi: 10.1111/j.1476-5381.2012.02211.x

Figure Lengend Snippet: Figure 7 Denosomin-mediated axonal outgrowth in primary cultures is mediated by vimentin secretion from astrocytes. (A–C) Isolated astrocytes were cultured in the presence or absence of 1 mM denosomin (Pre-Deno) for 6 days. Rat spinal cord cells were cocultured on the astrocyte layer without denosomin for 7 days after the astrocytes had been treated with denosomin. (D–F) Isolated astrocytes were cultured in the presence or absence of 1 mM denosomin (Pre-Deno) for 6 days. The culture medium was then replaced with fresh medium without denosomin, and the medium was collected 24 h later for use as astrocyte-conditioned medium (ACM). Rat primary cultured spinal cord cells were cultured in normal medium or ACM for 6 days (F). (G and H) Cultured astrocytes that were treated with or without denosomin (Deno) (G) and ACM collected from the astrocyte culture (H) were used for the ELISA assay. (I–K) Isolated mouse cortical cells were cultured with or without 1 or 10 ng·mL-1 vimentin for 6 days. The cells were cultured on slides with (J and K) or without (I–J) a CSPG coating. The cells were immunostained for pNF-H and MAP2, and the densities of pNF-H-positive axons on each neuron were quantified (B, D, I and K). Cellular distributions of neurons and non-neuronal cells per 0.1 mm2 were evaluated by the quantification of MAP2- and DAPI-positive cells (E). In (B, G and H): *P < 0.05, Student’s unpaired t-test (two-tailed); #P < 0.05; one-way ANOVA followed by the post hoc Bonferroni test, versus the control ACM treatment (D), versus the control cells (I) or versus CSPG-coated control cells (K). The numbers of photos (B, D, E, I, and K) or measurements (G and H) are shown in parentheses in the columns. The scale bar indicates 100 mm.

Article Snippet: The vimentin concentrations in these samples were quantified using a rat vimentin ELISA kit according to the manufacturer’s protocol (Cusabio Biotech, Wuhan, Hubei, China).

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Control