Journal: Frontiers in Oncology

Article Title: The FENDRR/FOXC2 Axis Contributes to Multidrug Resistance in Gastric Cancer and Correlates With Poor Prognosis

doi: 10.3389/fonc.2021.634579

Figure Lengend Snippet: FENDRR-regulated FOXC2 overexpression promotes GC drug resistance (A) qRT-PCR analysis of FENDRR, FOXF1, FOXL1 and FOXC2 expression in SGC7901/ADR cells infected with FENDRR shRNA or negative controls. (B) Western blot analysis of FOXC2 expression in SGC7901/ADR and SGC7901/VCR cells infected with FENDRR shRNA and in SGC7901 cells transfected with FENDRR vectors and the corresponding negative controls. (C) Expression levels of FENDRR in the nucleus and cytoplasm of the indicated cells was measured by qPCR. U1 and GAPDH were used as nuclear and cytoplasmic controls, respectively. (D) HEK-293 cells were transfected with FOXC2 luciferase reporter constructs and FENDRR-expressing vectors or shRNA and the corresponding negative controls. Luciferase activity values were measured and analyzed. Luciferase values are normalized to empty vector control values. (E) Survival of indicated cells after step-up concentration of ADR treatment for 72 h was evaluated using the CCK-8 assay. (F, G) Colony formation assay with the indicated cells treated with ADR (F) and the apoptotic rate of the indicated cells treated with 5-FU (G) are shown. (H, I) Colony formation assay (H) and apoptosis assay (I) of SGC7901/ADR and SGC7901/VCR cells infected with LV-shFENDRR with FOXC2 vector or control vector after ADR and 5-FU treatment, respectively. (J) LV-shFOXC2-infected cells were transplanted into the right flank of nude mice, and the corresponding control cells were transplanted into the left side. Left, representative images of tumors formed in nude mice (n=5) after ADR treatment are shown, scale bar, 1 cm. Middle, the tumor volumes of different groups were measured at the indicated time points. Right, the tumor weights of different groups measured after tumor isolation. (K) Quantification of the Ki67-positive staining of cells in xenografts from nude mice. ** P < 0.01, * P < 0.05, N.S. , not significant ( P > 0.05), error bars, s.d.

Article Snippet: Briefly, tissue sections were deparaffinized, subjected to antigen retrieval and endogenous peroxidase inactivation, and incubated with primary antibodies against FOXC2 (R&D Systems, AF6989) and Ki-67 (Abcam, ab15580).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Infection, shRNA, Western Blot, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Control, Concentration Assay, CCK-8 Assay, Colony Assay, Apoptosis Assay, Isolation, Staining

Journal: Frontiers in Oncology

Article Title: The FENDRR/FOXC2 Axis Contributes to Multidrug Resistance in Gastric Cancer and Correlates With Poor Prognosis

doi: 10.3389/fonc.2021.634579

Figure Lengend Snippet: FENDRR increases FOXC2 expression by competitively binding miR-4700-3p (A) Prediction of the potential miRNAs targeting both FENDRR and FOXC2 with two independent miRNA target databases. (B) qRT-PCR analysis of miR-4700-3p expression in the indicated cells. (C) The expression of miR-4700-3p following the downregulation or upregulation of FENDRR expression in the indicated cells. (D) Western blot analysis of FOXC2 expression in SGC7901/ADR and SGC7901/VCR cells transfected with miR-4700-3p mimics or inhibitors and the corresponding negative controls. (E, F) Predicted potential binding sites of miR-4700-3p to FENDRR and FOXC2 are shown (upper panel). The wild-type (wt) and mutant (mut) miR-4700-3p target sequences of FENDRR (E) and FOXC2 (F) were fused to a luciferase reporter and cotransfected into HEK-293 cells with miR-4700-3p mimics or a negative control. Luciferase activity values were measured and normalized to empty vector control values. (G, H) Western blot analysis of FOXC2 expression in the indicated cells. ** P < 0.01, * P < 0.05, N.S. , not significant ( P > 0.05), error bars, s.d.

Article Snippet: Briefly, tissue sections were deparaffinized, subjected to antigen retrieval and endogenous peroxidase inactivation, and incubated with primary antibodies against FOXC2 (R&D Systems, AF6989) and Ki-67 (Abcam, ab15580).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Western Blot, Transfection, Mutagenesis, Luciferase, Negative Control, Activity Assay, Plasmid Preparation, Control

Journal: Frontiers in Oncology

Article Title: The FENDRR/FOXC2 Axis Contributes to Multidrug Resistance in Gastric Cancer and Correlates With Poor Prognosis

doi: 10.3389/fonc.2021.634579

Figure Lengend Snippet: miR-4700-3p suppresses drug resistance in GC (A, B) Survival of SGC7901/ADR and SGC7901/VCR cells transfected with miR-4700-3p mimics (A) and SGC7901 cells transfected with miR-4700-3p inhibitors or the corresponding negative controls was evaluated using the CCK-8 assay after step-up concentration of ADR and 5-FU treatment for 72 h. (C, D) The apoptotic rate of the indicated cells treated with 5-FU was shown. (E, F) SGC7901/ADR and SGC7901/VCR cells were cotransfected with miR-4700-3p, FOXC2 vector or their negative controls. Cell survival (E) and apoptosis rate (F) of the indicated cells treated with ADR or 5-FU, respectively, was evaluated. (G) Representative data extracted from TCGA datasets showing correlation between FENDRR (left) and FOXC2 (right) expression with miR-4700-3p expression in GC tissues (n=100). (H) Kaplan-Meier analysis of the correlation between miR-4700-3p expression and overall survival in GC patients included in the TCGA datasets (n=100). ** P < 0.01, * P < 0.05, error bars, s.d.

Article Snippet: Briefly, tissue sections were deparaffinized, subjected to antigen retrieval and endogenous peroxidase inactivation, and incubated with primary antibodies against FOXC2 (R&D Systems, AF6989) and Ki-67 (Abcam, ab15580).

Techniques: Transfection, CCK-8 Assay, Concentration Assay, Plasmid Preparation, Expressing

Journal: Frontiers in Oncology

Article Title: The FENDRR/FOXC2 Axis Contributes to Multidrug Resistance in Gastric Cancer and Correlates With Poor Prognosis

doi: 10.3389/fonc.2021.634579

Figure Lengend Snippet: Positive correlation between FENDRR and FOXC2 expression in human GC tissue samples (A) Representative images of FENDRR and FOXC2 expression in 80 paired GC and adjacent normal tissue samples detected by FISH and IHC, respectively (left). Scale bar, 200 μm (low magnification) or 50 μm (high magnification). Analysis of immunohistochemical staining for FOXC2 in 80 paired CRC specimens and matched adjacent normal tissue samples (right). (B) Correlation between the expression of FENDRR and FOXC2 in 80 GC patients. (C) Kaplan-Meier analysis of the correlation between FOXC2 expression and overall survival in patients with GC. * P < 0.05, error bars, s.d.

Article Snippet: Briefly, tissue sections were deparaffinized, subjected to antigen retrieval and endogenous peroxidase inactivation, and incubated with primary antibodies against FOXC2 (R&D Systems, AF6989) and Ki-67 (Abcam, ab15580).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Oncology

Article Title: The FENDRR/FOXC2 Axis Contributes to Multidrug Resistance in Gastric Cancer and Correlates With Poor Prognosis

doi: 10.3389/fonc.2021.634579

Figure Lengend Snippet: Correlation of FOXC2 expression and patients’ clinicopathological variables in GC tissues.

Article Snippet: Briefly, tissue sections were deparaffinized, subjected to antigen retrieval and endogenous peroxidase inactivation, and incubated with primary antibodies against FOXC2 (R&D Systems, AF6989) and Ki-67 (Abcam, ab15580).

Techniques: Expressing

Journal: Stem Cells International

Article Title: Comparative Investigation of Human Amniotic Epithelial Cells and Mesenchymal Stem Cells for Application in Bone Tissue Engineering

doi: 10.1155/2015/565732

Figure Lengend Snippet: hAECs, hBMSCs, and hAFMSCs show a different molecular response to osteogenic induction in vitro . (a) Hierarchical cluster analysis of the differentially expressed genes involved in ossification. (b) Protein-protein interaction network analysis of the genes upregulated in hAECs, hBMSCs, and hAFMSCs after the 7-day osteogenic induction. (c) RUNX2, OSX, COLI, ALP, OPN, BMP6, FOXO1, and FOXC1 in osteogenic groups were gradually upregulated with time compared to those in the control group, while BMP4 and FOXC2 were only significantly upregulated in hAECs and hAFMSCs but not hBMSCs (∗: P < 0.05).

Article Snippet: Then all protein extracts were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, USA) and subjected to western blot with primary rabbit polyclonal antibody against FOXC2 (1 : 1000 Abcam, UK).

Techniques: In Vitro, Control

Journal: Stem Cells International

Article Title: Comparative Investigation of Human Amniotic Epithelial Cells and Mesenchymal Stem Cells for Application in Bone Tissue Engineering

doi: 10.1155/2015/565732

Figure Lengend Snippet: Verification of the upregulation of FOXC2 and ectopic osteogenesis of hAECs, hBMSCs, and hAFMSCs. (a) Expression of FOXC2 in all three cell types was significantly increased in a BMP2-dependent manner. (b, c) Both real-time PCR and western blot revealed that BMP2 significantly promoted the expression of FOXC2 in all the three cell sources. The western blot study also demonstrated that undifferentiated hBMSCs exhibited a higher expression level of FOXC2 than the other two cell sources. (d) Ectopic osteogenesis of hAECs, hBMSCs, and hAFMSCs in nude mice. β -TCP scaffolds carrying hAECs, hAFMSCs, and hBMSCs or alone were implanted subcutaneously for 4 weeks. HE staining showed no well-mineralized islands in either experimental groups or control group. Immunohistochemical staining showed that all cell types were viable as indicated by the positive expression of GFP. Moreover, OPN and OCN were evident in the experimental groups but not in the control group.

Article Snippet: Then all protein extracts were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, USA) and subjected to western blot with primary rabbit polyclonal antibody against FOXC2 (1 : 1000 Abcam, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Control, Immunohistochemical staining