Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A Heat maps of differentially expressed circRNAs in three ESCC tissues (T) and adjacent normal tissues (P). B Volcano plots showing changes in circRNA expression between cancerous and paracancerous tissues. C Schematic and Sanger sequencing of hsa_circ_0021727. D Agarose gel electrophoresis detection of hsa_circ_0021727 in KYSE510 cells. E The relative RNA levels were examined by qRT-PCR after treatment with RNase R or mock among total RNAs derived from KYSE 510 cells. F Random hexamer or oligo (dT)18 primers were utilized for reverse transcription assays. The relative RNA levels were examined using qRT-qPCR and normalized to those generated using random hexamer primers. G The subcellular localization of hsa_circ_0021727 in KYSE510 and TE-1 cells performed using FISH. H and I RNA-ISH detection of the expression of hsa_circ_0021727 in ESCC tissues and adjacent tissues. J Kaplan–Meier analysis of the OS in 162 patients with ESCC with high or low expression of hsa_circ_0021727. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing, Sequencing, Agarose Gel Electrophoresis, Quantitative RT-PCR, Derivative Assay, Random Hexamer, Reverse Transcription, Generated

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A and C qRT-PCR detection of the expression efficiency of hsa_circ_0021727 after cell transfection. B and D Wound healing assays for the migration ability of KYSE510 and TE-1 cells after cell transfection. E Transwell assays examining the invasive ability of KYSE510 and TE-1 cells after transfection of the hsa_circ_0021727 overexpression or knockdown vectors. F Three-dimensional (3D) spheroid invasion assays showing the effects of hsa_circ_0021727 on the growing antennae of KYSE510 and TE-1 cells. G and H MTT assays detecting the proliferation of KYSE510 and TE-1 cells after transfection of hsa_circ_0021727 overexpression or knockdown vectors. I Colony formation assays examining the proliferative capacity of KYSE510 and TE-1 cells transfected with hsa_circ_0021727 overexpression or knockdown lentiviral vectors. J EdU immunofluorescence assays detecting the proliferative capacity of KYSE510 and TE-1 cells. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Migration, Over Expression, Knockdown, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A Image suggesting that hsa_circ_0021727 might target bound miRNAs. B Probe efficiency for qRT-PCR detection. C and D Relative levels of five miRNAs in KYSE510 and TE-1 cells lysates pulled down by the hsa_circ_0021727 probe or the oligo probe. E and F qRT-PCR detection of the levels of five miRNAs in KYSE510 and TE-1 cells transfected with hsa_circ_0021727 overexpression or knockdown lentiviral vectors. G A luciferase reporter gene assay performed in KYSE510 and TE-1 cells co-transfected with hsa_circ_0021727 overexpression vector, pGL3 plasmid, pGL3-miR-23b-5p-wt, and pGL3-miR-23b-5p-mut. H FISH analysis showing the colocalization between hsa_circ_0021727 and miR-23b-5p in KYSE510 and TE-1 cells. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Quantitative RT-PCR, Transfection, Over Expression, Knockdown, Luciferase, Reporter Gene Assay, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A and B Wound healing assays evaluating the cell migration ability of KYSE510 cells and TE-1 cells co-transfected with miR-23b-5p and hsa_circ_0021727 overexpression vectors. C Transwell assays showing that miR-23b-5p and hsa_circ_0021727 jointly affected the invasion ability of KYSE510 and TE-1 cells. D Three-dimensional (3D) spheroid invasion assays showing the effects of hsa_circ_0021727 and miR-23b-5p on the growing antennae of KYSE510 and TE-1 cells. E and F MTT assays demonstrating the proliferation capacity of KYSE510 and TE-1 cells co-transfected with miR-23b-5p and hsa_circ_0021727 overexpression vectors. G Colony formation assays indicating that the cell proliferation capability of KYSE510 and TE-1 cells transfected with miR-23b-5p was reversed when the cells were co-transfected with hsa_circ_0021727 (values are expressed as the means ± SD; * P < 0.05).

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Migration, Transfection, Over Expression

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A Image showing the subcutaneous xenograft tumor tissues injected with KYSE510 cells co-transfected with miR-23b-5p and hsa_circ_0021727 overexpression vectors. B Graphs of tumor volumes in subcutaneous xenograft tumor models. C Image of lung metastatic nodules. Nude mice were injected with KYSE510 cells co-transfected with miR-23b-5p and hsa_circ_0021727 overexpression vectors. D Statistical analysis of lung metastatic nodules. E Lung tissue was stained using HE. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Injection, Transfection, Over Expression, Staining

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A Heatmap created from mRNA-seq (OE: overexpression, V: vector). B The top 20 enriched pathways in the mRNA data. C GSEA results showing that hsa_circ_0021727 correlated positively with NFκB signaling. D and E qRT-PCR detection of the expression of NFκB signaling-related factors in cells overexpressing or knocked down for hsa_circ_0021727. F Dual-luciferase assay detecting the luciferase activity related to NFκB signaling in cells overexpressing or knocked down for hsa_circ_0021727. G In cells overexpressing or knocked down for hsa_circ_0021727, western blotting was used to detect the protein levels of TAB1 and NFκB pathway factors. H TAB1 mutant and wild-type sequences were designed. I Dual-luciferase assays showing the luciferase activity after co-transfection of miR-23b-5p overexpression vector or NC vector, TAB1 wild-type or TAB1 mutant vector. J qRT-PCR and western blotting analyses of the expression of hsa_circ_0021727, miR-23b-5p, and TAB1 in ESCC patient tissues. K Analysis of the correlation between miR-23b-5p and TAB1. L Analysis of the correlation between hsa_circ_0021727 and TAB1. M In cells overexpressing hsa_circ_0021727 and knocked down for TAB1 , western blotting examined the protein level of TAB1 and downstream related factors. N In cells overexpressing hsa_circ_0021727 and miR-23b-5p, western blotting examined the protein level of TAB1 and downstream related factors. Western blot detection of the protein level of TAB1 and downstream related factors in KYSE510 and TE-1 cells overexpressing hsa_circ_0021727 and treated with INH14. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Over Expression, Plasmid Preparation, Quantitative RT-PCR, Expressing, Luciferase, Activity Assay, Western Blot, Mutagenesis, Cotransfection

Journal: The FASEB Journal

Article Title: Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation

doi: 10.1096/fj.201801422RR

Figure Lengend Snippet: LSD2 is essential for BAT differentiation in HB2 cells. A, B) Dox-inducible KD of LSD2. HB2 cells were transduced with tet-inducible shLSD2 (LSD2-KD#1 and #2) or control (shCtr) lentiviral vectors followed by blasticidin selection. Cells were cultured with or without Dox for 48 h. For relative mRNA levels of LSD2 (A), real-time quantitative PCR values were normalized to values for the 36B4 gene and are shown as fold differences against Dox (−). For levels of LSD2 protein (B), β-tubulin was used as a loading control. Values are mean ± sd (n = 3). *P < 0.05, †P < 0.01. C) Oil Red O staining of shRNA-expressing HB2 cells at d 7. Culture details are described in Supplemental Fig. S1A. Scale bars, 100 µm. D) Expression of adipogenesis and BAT-selective genes in shLSD2_#1 cells at indicated days. Quantitative RT-PCR values were normalized to those for the 36B4 gene and are shown as fold differences against Dox (−) at d 0. Values are mean ± sd (n = 3). *P < 0.05, †P < 0.01 vs. Dox (−) at each time point.

Article Snippet: Lentiviral expression of short hairpin RNA Lentiviral vectors for tetracycline (tet)-inducible short hairpin RNA (shRNA) expression were constructed using plasmids obtained from Riken BioResource Research Center (Saitama, Japan) according to the guidelines ( http://cfm.brc.riken.jp/Lentiviral_Vectors_J ).

Techniques: Transduction, Control, Selection, Cell Culture, Real-time Polymerase Chain Reaction, Staining, shRNA, Expressing, Quantitative RT-PCR