Journal: International Journal of Molecular Sciences

Article Title: Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells

doi: 10.3390/ijms21020527

Figure Lengend Snippet: Temporal redistribution of TJ proteins and barrier dysfunction caused by EPEC. ( A–C ) SKCO-15 cells were plated on Transwells and infected or not (UI) with EPEC. Total JAM-A, JAM-A S285, JAM-A Y280, occludin, and ZO-1 localization and TER were determined. ( A ) EPEC does not alter the distribution of total JAM-A. In contrast, JAM-A S285 is displaced from the cell–cell contacts to the cytoplasm at 30 min post-infection. Tyrosine phosphorylation of JAM-A Y280 is apparent at 60–120 min post-infection. Scale bars, 10 µm. ( B ) EPEC induces the endocytosis of occludin and ZO-1 at 1 and 2 h post-infection, respectively. Scale bars, 10 µm. ( C ) TER drops significantly as early as 45 min post-infection and progressively decreases over time as more TJ proteins are displaced. TER reported as percent change from baseline. * p < 0.01, *** p < 0.001.

Article Snippet: Par3 (07-330, EMD Millipore), Par6 (ab49776 and ab6022, Abcam, Cambridge, MA, USA), aPKCζ (sc-17781, Santa Cruz Biotechnology, Dallas, TX, USA), p-aPKCζ–T560 (ab62372, Abcam, Cambridge, MA, USA), p-aPKCζ–T410 (sc-12894R, Santa Cruz Biotechnology, Dallas, TX, USA), actin (A2066, Sigma-Aldrich, St. Louis, MO, USA), F-actin BODIPY 558/568 Phalloidin (B3475, Invitrogen, Life Technologies Carlsbad, CA, USA), occludin (33-1500, Invitrogen, Life Technologies, Carlsbad, CA, USA), JAM-A S285 (sc-17430, Santa Cruz Biotechnology, Dallas, TX, USA), JAM-A Y280 (600-401-GN5, Rockland Immunochemicals Inc, Limerick, PA), JAM-A (361700, Invitrogen, Life Technologies, Carlsbad, CA, USA), and ZO-1 (61-7300, Invitrogen, Life Technologies, Carlsbad, CA, USA).

Techniques: Infection, Phospho-proteomics

Journal: Frontiers in Oncology

Article Title: The Long Non-Coding RNA HOXC-AS3 Promotes Glioma Progression by Sponging miR-216 to Regulate F11R Expression

doi: 10.3389/fonc.2022.845009

Figure Lengend Snippet: | MiR-216 directly binds F11R to regulate the level of F11R. (A) 24 potential downstream genes of miR-216 were predicted using online databases. (B, C) The effect of miR-216 on the expression of candidate mRNAs in U87 and U251 cells was detected by qRT-PCR. (D) Binding sites of miR-216 on F11R. (E) Pearson correlation analysis showed that F11R was inversely correlated with miR-216 in glioma tissues. (F) The association between miR-216 and F11R was verified by luciferase reporter assay. (G) Expression of F11R in normal brain tissues and glioma tissues. (H) Expression of F11R in normal human astrocytes and glioma cell lines. (I) Expression of F11R in different grades of glioma identified using immunohistochemistry. (J) The expression levels of F11R in different tissues and cell lines were tested by western blot analysis. At the same time, it was verified that F11R was regulated by HOXC-AS3 and miR-216. Pearson’s correlation analysis for (E) ; t-test for (B, C, F, G) ; one-way ANOVA and post hoc test for (H) . **P < 0.01.

Article Snippet: Paraffin-embedded human and nude mouse glioma samples sections were incubated with primary antibodies against F11R (1:200, Proteintech, IL, USA) or Ki-67 (1:500 dilution, Servicebio, Wuhan, China) overnight at 4°C, and then incubated with the secondary antibody (1:200 dilution, Servicebio, Wuhan, China) at room temperature for 1 h. Next, the sections were stained with diaminobenzidine until brown precipitation appeared and counterstained with hematoxylin for 5 minutes.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Immunohistochemistry, Western Blot

Journal: Frontiers in Oncology

Article Title: The Long Non-Coding RNA HOXC-AS3 Promotes Glioma Progression by Sponging miR-216 to Regulate F11R Expression

doi: 10.3389/fonc.2022.845009

Figure Lengend Snippet: MiR-216 could inhibit glioma proliferation, migration, and invasion by targeting F11R. (A–C) qRT-PCR and western blot were used to test the expression of F11R in U87 and U251 cells transfected with NC, sh-F11R, miR-216 mimics, or miR-216 mimics together with F11R plasmids. (D, E) CCK-8 assay was used to test the proliferation of U87 and U251 cells transfected with NC, sh-F11R, miR-216 mimics, or miR-216 mimics together with F11R plasmids. (F–I) EdU assay was used to test the proliferation of U87 and U251 cells transfected with NC, sh-F11R, miR-216 mimics, or miR-216 mimics together with F11R plasmids. (J, K) Transwell assay was used to test the migration of U87 and U251 cells transfected with NC, sh-F11R, miR-216 mimics, or miR-216 mimics together with F11R plasmids. (L, M) Transwell assay was used to test the invasion of U87 and U251 cells transfected with NC, sh-F11R, miR-216 mimics, or miR-216 mimics together with F11R plasmids.One-way ANOVA and post hoc test for (A, B, G, I, K, M) . Two-way ANOVA and post hoc test for (D, E) . *P < 0.05, **P < 0.01.

Article Snippet: Paraffin-embedded human and nude mouse glioma samples sections were incubated with primary antibodies against F11R (1:200, Proteintech, IL, USA) or Ki-67 (1:500 dilution, Servicebio, Wuhan, China) overnight at 4°C, and then incubated with the secondary antibody (1:200 dilution, Servicebio, Wuhan, China) at room temperature for 1 h. Next, the sections were stained with diaminobenzidine until brown precipitation appeared and counterstained with hematoxylin for 5 minutes.

Techniques: Migration, Quantitative RT-PCR, Western Blot, Expressing, Transfection, CCK-8 Assay, EdU Assay, Transwell Assay

Journal: Frontiers in Oncology

Article Title: The Long Non-Coding RNA HOXC-AS3 Promotes Glioma Progression by Sponging miR-216 to Regulate F11R Expression

doi: 10.3389/fonc.2022.845009

Figure Lengend Snippet: HOXC-AS3 promoted the glioma malignancy phenotype by targeting the miR-216/F11R axis. (A–C) qRT-PCR and western blot were used to test the expression of F11R in U87 and U251 cells transfected with NC, sh-HOXC-AS3, or sh-HOXC-AS3 together with miR-216 inhibitors. (D, E) CCK-8 assay was used to test the proliferation of U87 and U251 cells transfected with NC, sh-HOXC-AS3, or sh-HOXC-AS3 together with miR-216 inhibitors. (F–I) EdU assay was used to test the proliferation of U87 and U251 cells transfected with NC, sh-HOXC-AS3, or sh-HOXC-AS3 together with miR-216 inhibitors. (J, K) Transwell assay was used to test the migration of U87 and U251 cells transfected with NC, sh-HOXC-AS3, or sh-HOXC-AS3 together with miR-216 inhibitors. (L, M) Transwell assay was used to test the invasion of U87 and U251 cells transfected with NC, sh-HOXC-AS3, or sh-HOXC-AS3 together with miR-216 inhibitors. *P < 0.05, **P < 0.01. One-way ANOVA and post hoc test for (A, B, G, I, K, M) . Two-way ANOVA and post hoc test for (D, E) . *P < 0.05, **P < 0.01.

Article Snippet: Paraffin-embedded human and nude mouse glioma samples sections were incubated with primary antibodies against F11R (1:200, Proteintech, IL, USA) or Ki-67 (1:500 dilution, Servicebio, Wuhan, China) overnight at 4°C, and then incubated with the secondary antibody (1:200 dilution, Servicebio, Wuhan, China) at room temperature for 1 h. Next, the sections were stained with diaminobenzidine until brown precipitation appeared and counterstained with hematoxylin for 5 minutes.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, CCK-8 Assay, EdU Assay, Transwell Assay, Migration

Journal: Frontiers in Oncology

Article Title: The Long Non-Coding RNA HOXC-AS3 Promotes Glioma Progression by Sponging miR-216 to Regulate F11R Expression

doi: 10.3389/fonc.2022.845009

Figure Lengend Snippet: HOXC-AS3 promotes glioma growth in vivo . (A) Luciferase signals were assessed at 7,14 and 21 days after implantation of glioma cells (6 mice per group). (B) Tumor maximum diameter was determined using H&E staining. (C) Overall survival of the sh-HOXC-AS3 and control groups was compared by Kaplan-Meier survival curves. (D) Ki-67 expression in the sh-HOXC-AS3 and sh-NC groups was measured by immunohistochemistry. (E) TUNEL staining was used to detect cell apoptosis in the sh-HOXC-AS3 and sh-NC groups. (F, G) Expression and quantification of miR-216 in the brain sections. (H, I) Expression and quantification of F11R in the brain sections. Log-rank test for (C) and t-test for (G, I) . *P < 0.05, **P < 0.01.

Article Snippet: Paraffin-embedded human and nude mouse glioma samples sections were incubated with primary antibodies against F11R (1:200, Proteintech, IL, USA) or Ki-67 (1:500 dilution, Servicebio, Wuhan, China) overnight at 4°C, and then incubated with the secondary antibody (1:200 dilution, Servicebio, Wuhan, China) at room temperature for 1 h. Next, the sections were stained with diaminobenzidine until brown precipitation appeared and counterstained with hematoxylin for 5 minutes.

Techniques: In Vivo, Luciferase, Staining, Control, Expressing, Immunohistochemistry, TUNEL Assay