Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a Schematic diagram illustrating the workflow for human CRISPR/Cas9 library screening. b The normalized read counts of all sgRNAs in CRISPR/Cas9 library plasmid ( n = 29,957) and HCCLM3 cells infected with human CRISPR/Cas9 library ( n = 29,875). The center line indicates the 50th percentiles ; bounds of box = 25th–75th percentiles ; whiskers = 10th–90th percentiles; minima: bottom whiskers; maxima: top whiskers. c Representative images of tumorspheres. large spheres (>70 µm); smaller spheres (<40 µm). d MAGeCK analysis and RRA ranking of top enriched genes in small tumorspheres compared to large tumorspheres. e Ranked dot plots of top enriched genes in small tumorspheres compared to large tumorspheres. P value was obtained by permutation test using Benjamini-Hochberg procedure by MAGeCK. f The normalized read counts of sgRNAs targeting SCARB2 between large tumorspheres and small tumorspheres. g Flow cytometric analysis of the proportion of CD24, EpCAM, CD13, or CD133 positive cells in CTRL Cas9 or SCARB2 Cas9 HCCLM3 cells. h Real-time PCR analysis of SCARB2 expression in CD133 + CD13 + and CD133 - CD13 - HCCLM3, HepG2 and primary HCC cells. i Cell growth curves of CD133 + CD13 + and CD133 - CD13 - HCCLM3 and HepG2 cells with or without SCARB2 deletion. j Representative images of tumorspheres of indicated cells with or without SCARB2 knockout. The number of spheres was counted. Scale bar, 50 μm. k Effect of SCARB2 depletion on sorafenib sensitivity in HCCLM3 and HepG2 cells. The data are a summary of IC 50 values for sorafenib. l Representative immunofluorescence images and quantification of the viability of HCC organoids with indicated treatment. Calcein acetoxymethyl (calcein-AM) was used to mark viable cells (green) and ethidium bromide homodimer-1 to mark dead cells (red) ( n = 10 organoids per group). Scale bar, 50 μm. m Relative cell viabilities of tumor organoids with or without SCARB2 knockout . n Representative images and quantification of protrusive invasion of HCC organoids. Scale bar, 50 μm. o Flow cytometric analysis of the proportion of CD133, CD13, EpCAM or CD24 positive cells in HCC organoids with or without SCARB2 knockout. p IHC staining of SCARB2 expression in human normal liver tissues and HCC specimens ( n = 90 per group). q Kaplan–Meier survival curves for patients with HCC stratified by SCARB2 expression. ( g , h , i , j , k , m , n , o ) n = 3 biological repeats. Statistical significance was calculated by ( g , h , i , j , l , m , n , o , p ) two-tailed Student’s t test; ( q ) two-sided log-rank test; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: For cell sorting, cell suspensions from HCC cell lines and primary human HCC cells were stained with PE anti-human CD13 and APC anti-human CD133, followed by being sorted CD133 + CD13 + and CD133 - CD13 - cells with FACS Aria III (BD Bioscience).

Techniques: CRISPR, Library Screening, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Expressing, Knock-Out, Immunofluorescence, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: a Scheme used to establish the model of spontaneous HCC with targeted Myc knock-in and Scarb2 knockout in the liver. b Representative photographs (top) and H&E staining (bottom) of intrahepatic tumor tissues in the indicated mice 8 weeks after birth. Scale bar, 1 cm. H&E staining Scale bar, 100 μm. c The liver weights of WT ( n = 12 mice), Cre Alb My c ( n = 12 mice), Cre Alb Scarb2 F/+ My c ( n = 12 mice) or Cre Alb Scarb2 F/F My c mice ( n = 6 mice). d Incidence of HCC in Cre Alb My c ( n = 11 mice), Cre Alb Scarb2 F/+ My c ( n = 12 mice) or Cre Alb Scarb2 F/F My c mice ( n = 10 mice). e Kaplan–Meier survival curves for Cre Alb My c ( n = 11 mice), Cre Alb Scarb2 F/+ My c ( n = 12 mice) or Cre Alb Scarb2 F/F My c mice ( n = 10 mice). f The tumor initiation efficiency of HCC cells harvested from indicated group was evaluated by in vivo limiting dilution assay ( n = 10 mice per group). g Flow cytometric analysis of the proportion of EpCAM, CD133 or CD24 positive cells in indicated group ( n = 3 mice per group). h Scheme used to establish DEN-induced HCC mouse model. i Representative photographs (top) and H&E staining (bottom) of intrahepatic tumor tissue in the indicated mice 8 months after birth. j Liver weights of the Cre Alb mice ( n = 8 mice) and Cre Alb Scarb2 F/F mice ( n = 8 mice) in DEN-induced HCC mouse model. k Numbers of tumor nodules in the indicated mice ( n = 8 mice per group). l Effect of SCARB2 knockout in HCCLM3 cells on tumor growth ( n = 8 mice per group). Representative images of tumors ( m ) and tumor weights ( n ) in the indicated group ( n = 8 mice per group). o–q Effects of SCARB2 knockout in HCCLM3 spheroids on tumor growth, tumor sizes and tumor weights ( n = 8 mice per group). r Flow cytometric analysis of the proportion of CD24, EpCAM, CD13, or CD133 positive cells in tumors generated from HCCLM3 spheroids with or without SCARB2 knockout ( n = 8 mice per group). Statistical significance was calculated by ( c , g , j , k , l , n , o , q , r ) two tailed Student’s t test; ( d, e ) two-sided log-rank test; ( f ) one-sided extreme limiting dilution analysis. Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: For cell sorting, cell suspensions from HCC cell lines and primary human HCC cells were stained with PE anti-human CD13 and APC anti-human CD133, followed by being sorted CD133 + CD13 + and CD133 - CD13 - cells with FACS Aria III (BD Bioscience).

Techniques: Knock-In, Knock-Out, Staining, In Vivo, Limiting Dilution Assay, Generated, Two Tailed Test

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: Primers used for real-time PCR

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques:

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: CIH promoted CSC-like properties in NSCLC cells. a Microscopic observation of NSCLC cells. A549 and SPCA1 spheroids were obtained and then cultured under Nor or CIH conditions for 24 h. b qRCP analyses in triplicate of CD44, Sox2, Nanog, Oct4, and CD133 in NSCLC cells. Adherent A549 and SPCA1 were treated with 24 h-Nor or CIH exposure. GADPH expression served as an internal control. c Flow cytometry analyses of the percentage of CD44 + CD133 + cells in A549 or SPCA1 populations. Error bars represent the mean ± SEM of at least triplicate experiments. * P < 0.05, ** P < 0.01. CIH chronic intermittent hypoxia, CSC cancer stem cell

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques: Cell Culture, Expressing, Flow Cytometry

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: Knockdown of Bach1 decreased the stemness and mitochondrial ROS accumulation in CIH-treated NSCLCs. Bach1 shRNA or parental negative control (NC) was transfected into A549 or SPCA1. Then the cells were cultured under CIH conditions for 48hrs. Flow cytometry analyses of CD44 + CD133 + cells in Nor or CIH-treated A549 ( a ) or SPCA1 ( b ). c The percentage of CD44 + CD133 + cells in Nor or CIH-treated cells was measured and illustrated. Fluorescence microscopy analysis for localization of mtROS production in A549 ( d ) and SPCA1 ( e ). Nuclei was stained with Hoechst (blue), mitochondria ROS were stained with MitoSOX-red. The merged panels showed the MitoSOX-red positive cells in total cells. f The percentage of MitoSOX positive cells was measured and illustrated. All experiments were performed in triplicate, and data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 compared with the control group

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques: shRNA, Negative Control, Transfection, Cell Culture, Flow Cytometry, Fluorescence, Microscopy, Staining