Journal: MedComm

Article Title: CircMALAT1 promotes cancer stem‐like properties and chemoresistance via regulating Musashi‐2/c‐Myc axis in esophageal squamous cell carcinoma

doi: 10.1002/mco2.612

Figure Lengend Snippet: Upregulation of circMALAT1 enhances CSC‐like features of ESCC cells in vitro and in vivo. (A) Expression of circMALAT1 in ESCC attached cells, spheroids, and spheroids‐reattached cells was examined by qPCR. (B) Representative images of ESCC spheroids generated from KYSE150/KYSE450 overexpressing or knockdown of circMALAT1 and corresponding control cells. Scale bar, 500 µm. (C) Western blot analysis of the stemness‐associated transcription factors (SOX2, OCT4, Nanog) in spheroids generated from KYSE150/KYSE450 overexpressing or knockdown of circMALAT1 and corresponding control cells. (D and E) Flow cytometric analysis of the CD133 and CD44 high /CD24 low expression level in spheroids generated from KYSE150 overexpressing circMALAT1 and corresponding control cells. (F–I) Animal models for the in vivo limiting dilution assay to assess the role of circMALAT1 in ESCC tumor initiation. (F) Schematic illustration of the experimental approach applied to assess the tumorigenicity and ESCC CSCs frequency in vivo. KYSE150 infected with lentivirus‐mediated Lenti‐vector and Lenti‐over‐circMALAT1 (left). NOD‐SCID mice were subcutaneously inoculated with ESCC cells dissociated from either KYSE150 vector spheroids or circMALAT1 spheroids and tumorigenicity was assessed two months after inoculation (right). (G) Tumor growth curve analysis of mice bearing Lenti‐over‐circMALAT1 spheroid cells (1 × 10 5 cells) compared with mice bearing control cells. (H) The images of the ultimately formed tumors (1 × 10 5 cells). (I) Representative images of immunohistochemical staining of CD90 and EpCAM in xenografted tumors. Scale bar, 100 µm. The data are presented as the mean ± SD, ** p < 0.01, *** p < 0.001.

Article Snippet: The flow cytometry test utilized the subsequent antibodies: PE anti‐human CD133 antibody, PE Mouse IgG1, κ isotype Control, FITC anti‐human CD24 antibody, FITC IgG2a, κ Isotype Control (BioLegend, USA), APC anti‐human CD44, APC IgG2b Isotype Control (Proteintech, USA).

Techniques: In Vitro, In Vivo, Expressing, Generated, Knockdown, Control, Western Blot, Limiting Dilution Assay, Infection, Plasmid Preparation, Immunohistochemical staining, Staining

Journal: MedComm

Article Title: CircMALAT1 promotes cancer stem‐like properties and chemoresistance via regulating Musashi‐2/c‐Myc axis in esophageal squamous cell carcinoma

doi: 10.1002/mco2.612

Figure Lengend Snippet: CircMALAT1 enhances MSI2 stability by disrupting the interactions between MSI2 and BTRC E3 ubiquitin ligase. (A) LC–MS/MS were performed to identify circMALAT1 interacting proteins in KYSE150 after pull‐down with biotinylated circMALAT1 probe and control (left). Correlation between reported ESCC CSC markers (SOX2, OCT4, NANOG, ABCG2, CD90, BMI1, CD271, CD44, and CD133) and mRNA level of candidate protein interaction with circMALAT1(Fold change of probe/control ≥ 5.0) in ESCC in TCGA database. (B) RNA immunoprecipitation (RIP) assay in KYSE150 confirmed that circMALAT1 could be enriched by MSI2. LncRNA‐NEAT1 was used as the negative control. (C) The interaction between circMALAT1 and MSI2 was confirmed by RNA pull‐down assays and Western blot analysis. (D) Colocalization analysis of circMALAT1 and MSI2 using protein IF and RNA FISH assays, respectively. Scale bar, 30 µm. (E) Western blot detection of MSI2 protein half‐life in KYSE150 cells transfected with the circMALAT1 and indicated control and treated with cycloheximide (CHX) (100 µg/mL) for the indicated timepoints. (F) Western blot of MSI2 levels in the circMALAT1‐siRNA cells and corresponding control cells were treated with proteasome inhibitor MG132 (10 µM) or chloroquine (10 µM) for 12 h. (G) IP was performed with anti‐HA antibody in KYSE150 cells transfected with HA‐BTRC and Flag‐MSI2 plasmids in the presence or absence of circMALAT1, followed by immunoblotting (IB) with the indicated antibodies. The cells were treated with MG132 (10 µM) for 12 h before harvesting. (H) KYSE150 cells were cotransfected with the indicated plasmids and treated with MG132 (10 µM) for 12 h before collection. MSI2 pull‐down experiments were conducted, and the samples were analyzed using western blotting with the indicated antibodies. The data are presented as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The flow cytometry test utilized the subsequent antibodies: PE anti‐human CD133 antibody, PE Mouse IgG1, κ isotype Control, FITC anti‐human CD24 antibody, FITC IgG2a, κ Isotype Control (BioLegend, USA), APC anti‐human CD44, APC IgG2b Isotype Control (Proteintech, USA).

Techniques: Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Control, RNA Immunoprecipitation, Negative Control, Western Blot, Transfection