Fig 2: Sequence comparisons of thalidomide-related regions in vertebrate PLZF, SALL4 and CRBN Alignment of amino acid sequence of ZNF1 and ZNF3 in PLZF among human, rabbit, mouse, chicken and zebrafish. The red letters indicate different sequences of ZNF1,3 in PLZF among the species. The white letters indicate critical Gly residue for interaction between PLZF and CRBN with thalidomide or its derivatives.Alignment of amino acid sequence of ZNF2 in SALL4 among the species above. The red letters indicate different sequences of ZNF2 in SALL4 among the species. The white letters indicate critical Gly residues for interaction between SALL4 and CRBN with thalidomide or its derivatives.Alignment of amino acid sequence in CRBN among the species above. The blue and red letters indicate different sequences of thalidomide-binding region in CRBN among the species.

Fig 3: Effects of SALL4 ZFC1 and ZFC2 deletion in ESCs on chromatin binding, gene expression, and differentiation(A) Diagram showing the in-frame deletion within the Sall4 coding sequence, generated by CRISPR-Cas9.(B) SALL4 ZFC1-2? localization determined by immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars, 3 µm.(C) Heatmap and profile plot showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines.(D) Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit ±250 bp) in WT (blue) and ZFC1-2? (purple) ESCs.(E) Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between ZFC4mut and ZFC1-2? ESCs. ZFC4-regulated genes are indicated in red and ZFC1/2-regulated genes in purple.(F) Correlation between gene mis-regulation (log2 fold change versus WT) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes were divided into five equal categories depending on their AT content.(G) TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars, 100 µm.See also Figure S6 and Table S2.

Fig 4: SALL4 promotes the proliferation of cervical cancer cells in vitro. A, Immunocytochemical staining showing SALL4 expression in HeLa, SiHa, C33A and CaSki cells; original magnification, ×1000. B, Representative western blots are shown of the SALL4 expression in HeLa, SiHa, C33A and CaSki cells. SALL4‐modified cervical cancer cell lines were identified by western blot: C, control (HeLa‐GFP) and SALL4‐overexpressing HeLa (HeLa‐SALL4) cells; D, control (SiHa‐GFP) and SALL4‐overexpressing SiHa (SiHa‐SALL4) cells; E, control (C33A‐shNC) and SALL4‐silenced C33A (C33A‐shSALL4) cells. The growth and viability of cervical cancer cells were detected using growth curves and the 3‐(4,5‐dimethylthiazole‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay in HeLa‐GFP and HeLa‐SALL4 cells (F, H), SiHa‐GFP and SiHa‐SALL4 cells (G, I), and C33A‐shNC and C33A‐shSALL4 cells (J, K). Values are shown as the mean ± SD obtained from three separate experiments. *P < .05 vs control, **P < .01 vs control

Fig 5: Correlations among SALL4, miR-200c, and PD-L1 in the center tumor regions of HCC. a, c Immunostaining of SALL4 and PD-L1 proteins and miR-200c in situ hybridization in the center tumor a and peritumor c regions of three representative HCC patients. Original magnifications: × 10, × 20; bar = 100 µm, 50 µm, respectively. b, d Pearson correlation analysis between the expression (IOD/area) of SALL4, PD-L1 and miR-200c in center tumor (b) and peritumor (d) regions of HCC. e Correlation analysis between SALL4 and miR-200c (Relative ΔCt) (left), SALL4 and PD-L1 (middle), and PD-L1 and miR-200c (right), from eight fresh tissue HCC samples. Spearman’s correlation coefficients and P values are shown