Journal: Journal of Translational Medicine

Article Title: CircNDC80 promotes glioblastoma multiforme tumorigenesis via the miR-139-5p/ECE1 pathway

doi: 10.1186/s12967-022-03852-3

Figure Lengend Snippet: cNDC80 induces GBM cell proliferation, migration, and invasion in vitro. A The expression levels of cNDC80 in GBM cells and NHAs were investigated using qRT–PCR. B Designated shRNAs for cNDC80 are shown schematically at splice junctions. C In U87 and pGBM-1 cells transduced with either cNDC80-targeting shRNA-3 or a non-targeted control shRNA, the expression of cNDC80 was measured by qRT–PCR. D – F CCK-8, colony formation, and EdU were used to identify the cell proliferation capacities of transfected U87 and pGBM-1 cells. G Transwell for cell migration and invasion studies after expression vector or shRNA transfection of glioma cells. H Using the wound healing assay, the migration of GBM cells was evaluated. I Following transfection in GBM cells, N-cadherin, Vimentin, and CyclinD1 were analyzed using Western blots. Each experiment was conducted three times, and the findings are shown as mean ± SD. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Article Snippet: Subsequently, primary antibodies were added at 4℃: anti-ECE1 (abcam, #ab71829,1:1000), anti-Sox2 (#ab97959); Cell Signaling Technology: anti-N-cadherin (# 13116,1: 1000), anti-Vimentin (# 5741, 1:1000), anti-CyclinD1 (# 55506, 1:1000), anti-Oct4 (# 2750, 1:1000), anti-Nanog (#4903, 1:1000), anti-GAPDH (#5174,1:1000) was used overnight.

Techniques: Migration, In Vitro, Expressing, Quantitative RT-PCR, Transduction, shRNA, CCK-8 Assay, Transfection, Plasmid Preparation, Wound Healing Assay, Western Blot

Journal: Theranostics

Article Title: KLF5 inhibition potentiates anti-PD1 efficacy by enhancing CD8 + T-cell-dependent antitumor immunity

doi: 10.7150/thno.82182

Figure Lengend Snippet: Klf5 is necessary for tumor-intrinsic PGE2 generation. ( A ) Heatmap of differentially expressed genes involved in arachidonic acid metabolism from both Klf5-deficient or control EMT6 and Klf5-overexpressing or control 67NR tumors. ( B ) Relative Ptgs2 (encoded COX2) mRNA expression in control and Klf5-deficient EMT6 cells in the presence or absence of LPS (1 μg/mL) or celecoxib (CEL) (50 μM). ( C ) Relative Ptgs2 (encoded COX2) mRNA expression in control and Klf5-overexpressing 67NR cells treated with or without LPS (1 μg/mL) or celecoxib (CEL) (50 μM). ( D ) Western blot analysis of Klf5, CyclinD1 and Cox2 in both Klf5-deficient or control EMT6 and Klf5-overexpressing or control 67NR cells following treatment with or without LPS (1 μg/mL) or CEL (50 μM) for 24 h. ( E ) HEK293T cells were cotransfected with Ptgs2 (-1000/+101)-luc or Ptgs2mut (-1000/+101)- luc plus GV341-KLF5 or control vector GV341 and the internal control plasmid pRL-TK. ( F-G ) 67NR wt/Klf5-3F-OV cells were subjected to ChIP assays using anti-Flag magnetic beads. PCR was performed to amplify regions surrounding the putative Klf5 binding region and a nonspecific Klf5 binding region. ( H-I ) The secreted levels of PGE2 were detected by ELISA in both Klf5-deficient or control EMT6 and Klf5-overexpressing or control 67NR cells following treatment with or without LPS (1 μg/mL) or CEL (50 μM) for 24 h. ( J ) Pge2 levels were evaluated in Klf5-deficient versus control EMT6 tumors receiving daily CEL (30 mg/kg) treatment for 1 week (n = 3). ( K-M ) The Klf5, Cox2 and Cd8 levels were quantified by ImageJ after staining with specific antibodies in paraffin-embedded tissues obtained from Klf5-deficient versus control EMT6 tumors. Representative images of Klf5, Cox2 and Cd8 ( K ). Cox2 expression was quantified in ( L ). The level of Cd8 + cells was quantified in ( M ). Scale bar equals 50 μm. The data are represented as means ± SD. n ≥ 5 for mice in each group. (*p < 0.05; **, p < 0.01; ***, p < 0.001 significant vs. control; one-way or two-way ANOVA).

Article Snippet: The membranes were blocked with 0.05% Tween 20 (#P9416; Sigma Aldrich) v:v in Tris-buffered saline (TBS) (TBST) (#ET220; Euromedex) supplemented with 5% nonfat powdered milk (w:v in TBS), followed by overnight incubation at 4 °C with primary antibodies specific for KLF5 (#AF3758; 1:1000; R&D Systems), COX2 (#66351-1-Ig; 1:1000; Proteintech), CyclinD1 (#55506; 1:1000; Cell Signaling Technology) and Vinculin (#13901; 1:2000; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Plasmid Preparation, Magnetic Beads, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining