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Sox9 Controls Self-Renewal of Oncogene Targeted Cells and Links Tumor Initiation and Invasion

http://www.cell.com/cell-stem-cell/abstract/S1934-5909(15)00217-9?rss=yes by

 

Figure 6

Sox9 Directly Controls ECM, Adhesion, and Cytoskeleton Remodeling during Tumorigenesis

(A) Sox9 ChIP-seq peaks associated with Emb, Lama3, and Lamb3. Red bar denotes MACS peak position; gene is represented in light blue, and boxes represent exons. Arrow represents transcription orientation.

(B) PCR quantification of Sox9 ChIP in the regulatory regions of Lamb3, Lama3, Col4a3, Mcam, and Emb. Enrichment is normalized over control IgG. PCR primers are designed within the peak and 2 kb upstream of the peak for negative controls.

(C) qRT-PCR analysis of cell adhesion and ECM and cell adhesion genes in FACS-isolated cells from K14CreER:SmoM2 mice and in K14CreER:SmoM2:Sox9cKO mice 4 and 9 weeks after TAM administration. Fold changes were calculated over K14CreER:SmoM2 mice 4 weeks after TAM using the delta delta CT method after normalization over TBP. Data represent the mean and SEM of at least three biological replicates.

(D) SHG analyzed by multiphoton confocal microscopy in WT, K14CreER:SmoM2, and K14CreER:SmoM2:Sox9cKO mice 4, 6, and 9 weeks after TAM administration.

(E and F) Immunostaining of (E) α6 integrin and (F) Mcam and SmoM2 in K14CreER:SmoM2 mice and in K14CreER:SmoM2:Sox9cKO mice 4, 6, and 9 weeks after TAM administration.

(G) EM analysis of WT, K14CreER:SmoM2, and K14CreE:SmoM2:Sox9cKO mice 9 weeks after TAM administration. BC, basal keratinocytes; D, dermis.

(H) Sox9 peaks associated with Acf7 and Palld.

(I) PCR quantification of Sox9 ChIP in the regulatory regions of Palld and Acf7. Enrichment is normalized over control IgG.

(J) Relative mRNA expression of actin cytoskeleton regulators in FACS-isolated cells from K14CreER:SmoM2 mice and in K14CreER:SmoM2:Sox9cKO mice 4 and 9 weeks after TAM administration. Data are normalized over K14CreER:SmoM2 mice 4 weeks after TAM administration and represent the mean and SEM of at least three biological replicates.

(K) IF of filamentary actin (F-actin) stained with phalloidin in K14CreER:SmoM2 and K14CreER:SmoM2:Sox9cKO mice.

Exposure time is 50 ms for all acquisition. The scale bars represent 50 μm, except in (G), where it represents 100 nm. Data represent the mean and SEM of at least three biological replicates. p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.