Fig 2: JIB-04 impairs the maintenance and viability of cancer stem-like cells in HCC cells. (A) CD133+/CD13+ subpopulations in PLC/PRF/5 cells after treatment with DMSO or 6 µM JIB-04 for 24 h were analyzed with flow cytometry analysis. Treatment with JIB-04 resulted in a decreased percentage of CD133+/CD13+ subpopulations compared with DMSO (control). Data are mean ± SEM of triplicate measurements; * p <0.05, ** p < 0.01, and *** p < 0.001. (B) Phase contrast image of tumorspheres derived from cells cultured with DMSO (control), 6µM JIB-04, or 4µM TSA for 24 h (Left panel). Percentage of sphere-initiating cells measured using CCK assay (Right panel). The number of DMSO-treated cells was set as 100 (n = 3). The data indicated that JIB-04 reduces tumor-initiating ability in HCC cells. (C) Phase contrast image of primary tumorspheres after treatment with DMSO (control), 6µM JIB-04, or 4µM TSA for 2 days (Left panel). Viability of primary tumorspheres measured using CCK assay (Right panel). The number of DMSO-treated cells was set as 100 (n = 3). (D) Phase contrast image of secondary tumorspheres developed from primary tumorspheres without drug treatment for 12 days. Viability of secondary tumorspheres measured with CCK assay (Right panel). The number of DMSO-treated cells was set as 100 (n = 3). (E) Western blot analysis of histone methylation levels after treatment with DMSO or 6µM JIB-04 for 24 h. H3 was used as a loading control. (F) The tumor-initiating ability of CSCs in PLC/PRF/5 cells was decreased due to the deficiency in KDM4B, KDM4D, and KDM6B. Phase contrast image of tumorspheres derived from KDM4B-, KDM4D-, or KDM6B-depleted knockdown cells (Left panel). Percentage of sphere-initiating cells measured with CCK assay (Right panel). The number of DMSO-treated cells was set as 100 (n = 3). Data are represented as mean ± SEM of triplicate measurements; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Fig 3: Knockdown of ABI2 inhibits HCC malignant behaviour and cancer stem cell properties, which can be rescued by MEOX2 overexpression. (A, B) Summary graph showing the HCC cell 2D growth rate. (C, D) Representative images of foci formation. (E, F) Transwell assay of HCC cells in PLC/PRF/5 and Huh7 cells. (G, I). Flow cytometric analysis of CD24+, CD133+ and CD326+ cancer stem cell properties in PLC/PRF/5 and Huh7 cells. (J, K). Sphere formation assays were performed to investigate HCC cancer stem cell properties in PLC/PRF/5 and Huh7 cells. In all panels, data are presented as the mean ± SD. (*p < .05, **p < .01, ***p < .001, ns: no statistical significance).

Fig 4: Neuroblastoma stem cells have highly enriched GPC2 expression(A and B) GPC2 histograms (A) and GPC2 versus CD133, CD338, CD117, and GD2 flow cytometry plots (B) of neuroblastoma PDXs/xenografts.(C) Fold-change GPC2 MFI plot of GPC2UltraHi versus GPC2Hi cells in neuroblastoma PDXs/xenografts.(D) Fold-change stem cell marker MFI plot of GPC2UltraHi versus GPC2Hi cells in neuroblastoma PDXs/xenografts.(E and F) Gpc2/GPC2 histograms (E) and Gpc2/GPC2 versus CD133, CD338, CD117, and GD2 flow cytometry plots (F) of murine (9464D) and human (SK-N-AS) neuroblastoma cell lines.(G) Fold-change stem cell marker MFI plot of GPC2UltraHi versus GPC2Hi cells in neuroblastoma cell lines.(H) GPC2 (left) and CD133 (right) histograms in SK-N-AS cells after D3-GPC2-PBD ADC treatment.(I) Quantification of CD133- and GPC2-positive SK-N-AS cells after D3-GPC2-PBD ADC treatment (left, ADC × 1; right, ADC × 2).(J) GPC2 western blot of SK-N-SH/NBL-SRPMI/NB cells.(K) Relative growth of SK-N-SH/NBL-SRPMI/NB cells 4 days after treatment with the D3-GPC2-PBD ADC.(L) Fold-change MFI plot of SH-SY5Y-EmptyNB and -GPC2RPMI/NB isogenic cells.Data in (C) and (D) are represented as mean ± SEM of PDXs/xenografts analyzed 2–3 independent times with 4 technical replicates each time for each PDX/xenograft in (C). Data in (G), (I), and (L) are represented as mean ± SEM of experiments done 2–3, 2, and 5 times, respectively. Data in (K) represent mean ± SEM from a representative experiment done in technical triplicate at least two independent times.Neg, negative.*p < 0.05; **p < 0.001; ***p < 0.0001. See also Figure S2.

Fig 5: Knockdown of ABI2 inhibits HCC cell growth, sorafenib resistance, migration, invasion and cancer stem cells expansion. (A, B) shRNA against ABI2 significantly down-regulated the expression of ABI2 in PLC/PRF/5 and Huh7 cells detected with western blotting and Quantitative RT-PCR. Scrambled shRNA was used as negative control. (C, E) Summary graph showing the HCC cell 2D growth rate. (D, F) Summary graph showing the PLC/PRF/5 and Huh7 cells 2D growth rate in 10, 7 and 5µM sorafenib. (G, H) Foci formation assays were performed in HCC cell lines to evaluate cell proliferation ability. (I, J) Down-regulated ABI2 expression could inhibit cell migration and invasion in PLC/PRF/5 and Huh7 cells. (K–M) Flow cytometric analysis of CD24+, CD133+ and CD326+ cell properties in PLC/PRF/5 and Huh7 cells. (N) Sphere formation assays were performed to investigate HCC cancer stem cell properties in PLC/PRF/5 and Huh7 cells. In all panels, data are presented as the mean ± SD. (*p < .05, **p < .01, ***p < .001, ns: no statistical significance).