Fig 1: Cell viability and transcriptomic changes after knockdown of SOX11. (a) Expression level of SOX11 after knockdown, as analyzed by RT-qPCR (N = 3, black lines indicate the median). (b) Knockdown of SOX11 caused no evident changes in cell viability. Curves are drawn from the biological replicates using the median value at given time points. 697 and RCH-ACV cells represent the T/P subtype, and REH cells represent the E/R subtype. SOX11 knockdown was confirmed by Western blotting, and cell viability assessments were conducted with the AlamarBlue assay. Measured absorbance intensities are reported as x103 (697 N = 6; RCH-ACV and REH N = 4). Western blot gel figures are cropped per cell line from the original blot figures shown in Fig. S3. (c) SOX11 knockdown in 697 cells caused downregulation of genes that are known to be altered after MYC upregulation. (d) Venn diagram of differentially expressed genes in the REH, 697, and RCH-ACV cell lines after knockdown of SOX11 (adjusted p-value < 0.05). (e) Heatmap of 15 concordantly differentially expressed genes in all three cell lines after knockdown of SOX11. Reproduced with permission66.
Fig 2: Fscn1 is regulated by SOX11 and SOX4 during development and wounding (a, b) Immunohistochemical analysis of FSCN1 expression in WT skin during development (a), in dcKO skin at E14.5 (b) and skin with FLAG-SOX11 induced since birth at P5 (c). d RT-qPCR analysis of Fscn1 expression in E16.5 epidermis and primary keratinocytes with the indicated genotypes. Data are the mean ± SD. n = 3 biological replicates. e Immunofluorescence analysis of FSCN1 expression in keratinocytes of specified genotypes, with FSCN1 localized at cell edges (arrowed). f Immunohistochemical analysis of FSCN1 expression in WT and dcKO skin 5 days post wounding. g Immunofluorescence analysis of filopodia with anti-VCL (vinculin) and iFluor 647-conjugated phalloidin (to detect actin filaments). h Graph quantifying the filopodia length in keratinocytes. Data are the mean ± SD. n = 80 (WT), 63 (Sox4 cKO), 83 (Sox11 cKO), or 88 (dcKO). ***p < 0.001 (Student’s two-tailed t-test). i Western analysis of WT or dcKO keratinocytes expressing FLAG-tagged CRISPR/Cas9 and control gRNAs or gRNAs targeting Fscn1. The representative blots from two independent experiments. j Graph quantifying relative areas the cells migrated normalized over the controls (WT cells expressing control gRNAs). n = 3 biological replicates, with 12–18 fields quantified per replicate. Data are the mean ± SD. ***p < 0.001 (Student’s two-tailed t-test). k Immunofluorescence analysis of FLAG-FSCN1 expression in FACS-isolated GFP + cells from keratinocytes that were transduced to express GFP with tet-inducible FLAG-FSCN1 and with or without 24 h Dox treatment. l WT or dcKO keratinocytes were transduced to express GFP alone or GFP with FLAG-FSCN1. Graph quantifying relative areas the cells migrated normalized over the control (WT cells with empty vector without Dox). n = 3 independent experiments with 12–18 fields quantified per replicate. Data are the mean ± SD. ***p < 0.001 (Student’s two-tailed t-test). Epi epidermis, der dermis, HF hair follicles, Es eschar; LE leading edge, GL granulation layer. Scale bars, 20 µm (a–c, k), 100 µm (e), 50 µm (f), 10 µm (g). Images are representative of 2 (e, k) or 3 (a–c and f) biological replicates. Source data for panels d, h–j and l are provided as a Source Data file
Fig 3: SOX11 overexpression induces embryonic transcriptional program. a Heat map from two-color microarray analysis depicts clustering of differentially expressed (FDR < 0.05 and fold change > 1.5) probesets in K14-rtTA;TRE-Sox11 epidermis relative to control K14-rtTA epidermis at P4 after 12 h of Dox induction. Data from two biological replicates of each genotype. b Top ten GO biological processes of the differentially expressed genes in SOX11-induced epidermis. Cell organization and movement related (blue) and epidermal differentiation (red) processes are among the most significantly enriched. c High overlap of SOX11-induced signature genes and embryonic epidermal signature genes. The Venn diagrams show overlapping between E13.5 epidermal signature genes and genes changed by SOX11-induction (genes with log2-fold change > 1.5 in both microarrays). Venn diagram hypergeometric p values and the enrichment level (R) of the overlaps are indicated below each Venn diagram, with statistically significant values highlighted in red. d Percentage of genes altered by SOX11 overexpression overlapping with embryonic signature genes. Source data for panels a, c and d are provided as a Source Data file
Fig 4: Histological images of lymphoma samples from the (A) spleen and (B) duodenum stained with (a, b) hematoxylin and eosin, (c) CD20 (L26; DAKO, Carpinteria, CA), (d) CD5 (4C7; Leica, Wetzler, Germany), (e) cyclin D1 (SP4-R; Roche, Mannheim, Germany), (f) Sox11 (polyclonal, HPA000536; Sigma-Aldrich, St Louis, MO), and (g) Ki-67 (MIB-1; DAKO). Original magnification, (a) ×40; (b–g), ×400.
Fig 5: Identifying downstream effectors of SOX11 and SOX4 in embryonic epidermis. a qRT-PCR analysis of isolated epidermis from Sox11 cKO, Sox4 cKO, or dcKO and their WT littermate embryos at E16.5 with two biological replicates per genotype. The values are gene expression levels in the knockouts relative to their respective wild-type controls. Data are the mean ± SD. n = 2 biological replicates. b Two-color microarray analysis was performed on E16.5 epidermis lacking either Sox11 or Sox4, or both genes, as well as on wild-type littermate, which is used as a baseline control. Heat map shows clustering of probesets with significant differential expression (FDR < 0.05 and fold change > 1.5) in dcKO, Sox11 cKO or Sox4 cKO epidermis relative to their WT littermate controls. n = 2 biological replicates. c Venn diagram showing the overlap of probesets significantly changed in dcKO, Sox11 cKO or Sox4 cKO epidermis. d Cell organization/motility (blue) and epidermal development (red) are among the top GO biological processes found in the differentially expressed genes in dcKO epidermis at E16.5 (as determined by Metascape, q values < 0.05). e Venn diagrams (Top panel) show the overlaps of genes up- or downregulated (log2-fold change ≥ 1.5 and FDR < 0.05) in E16.5 dcKO epidermis with genes up- or downregulated in E13.5 epidermal cells as compared to P4 basal epidermal cells. Hypergeometric p values and the enrichment level (R) of the overlap show statistical significance between specified groups (highlighted in red). f Graph shows the percentage of genes altered in dcKO overlapping with embryonic signature genes. Source data are provided as a Source Data file
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