(A) In the absence of Notch1, only very few ETPs can be isolated for the detection of T cell lineage precursors

The FACS plots are gated on Lin CD44 CD25 DN1 thymocytes derived from Notch1//CCR9/+ and Notch1//MxCre//CCR9/+ mice 2 wk after pIpC injection. Percentages are indicated. (B) Single TMPs were sorted from Notch1//CCR9/+ and Notch1//MxCre//CCR9/+ mice 4 wk after pIpC injection on OP9-DL1 stromal layers and cultured for 12–14 d. Cells falling into the right half of the ETP gate shown in A were considered TMPs. DN3/4 thymocytes developed on OP9-DL1 stroma even in the absence of Notch1, as previously described (reference ). Percentages are indicated. (C) Genomic PCR was performed on DNA purified from the OP9-DL1 culture of DN3/4 thymocytes generated from a single TMP of a Notch1//CCR9/+ and a Notch1//MxCre//CCR9/+ mouse 4 wk after pIpC injection. Two sets of primers were used, with one detecting the wild-type (N1 wild-type) and the floxed (N1 allele) Notch1 allele (left lane) and the other detecting the deleted allele of Notch1 (Δ N1; right lane). Because the added hematopoietic cells did not carry a wild-type Notch1 allele, this band is thought to be derived from OP9-DL1 stroma. PCR fragment sizes in base pairs are given in parenthesis.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p

(A) LSKs were fractionated according to their Flt3 and CCR9-EGFP expression

The FACS plot on the left is gated on Lin cells, and the plot on the right is gated on LSK cells (as shown in the plot on the left). LSK subpopulations were sorted from the indicated regions. Percentages are indicated. (B) 100 cells of the indicated precursors were sorted onto OP9-DL1 stroma, cultured for 9 d, and analyzed by FACS. The levels of CCR9-EGFP and CD44 expression are indicative of the maturity of each precursor, as T lineage–biased precursors are expected to progress faster, whereas precursors without a T lineage bias or those biased toward a non-T fate will take longer to develop to DN3 thymocytes. The histogram plots are gated on DN2/3 thymocytes in which CCR9-EGFP expression increases with the progression from DN2 to DN3 thymocytes (reference ). The results of three independent wells are shown in each histogram plot. Contour plots are gated on Lin CD90 cells, and the numbers in each quadrant indicate the mean frequency of each population from three independent experiments. (C) The results of a representative clonogenic myeloid progenitor assay are shown for a 96-well plate seeded with 5, 10, 20, or 40 cells of the indicated precursors. The number of detectable myeloid colonies for each well is given. (D) Semiquantitative RT-PCR was performed on fivefold dilutions of cDNA prepared from the indicated precursors with primers specific for the indicated transcripts. Primers specific for hypoxanthin-phosphoribosyl-transferase (HPRT) transcript were used as a loading control. PCR fragment sizes in base pairs are given in parenthesis.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p

The LSK population was analyzed by FACS in CCR9/ //Flt3∷Cre//R26R-EYFP mice

The top left plot is gated on LSK bone marrow cells, and the histogram plots are gated on the indicated regions of interest. The fraction of EYFP, recombined cells is shown for each subset. Percentages are indicated.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p

(A) Lin blood cells of heterozygous CCR9-EGFP knock-in mice were fractionated according to their CD117 and CCR9 expression

The plot is gated on Lin cells. Percentages are indicated. (B) Lin fractions with the indicated phenotypes were isolated from blood (as shown in A) and from bone marrow, and were cultured as groups of 100 cells on OP9-DL1 stromal layers for 12 and 19 d. The fraction of wells that contained T lineage–committed DN3/4 thymocytes is plotted for each time point. At least 1,200 cells cultured as 100 cells per well were assayed for each population and time point. For the analysis of T lineage precursor activity of Lin CD117 CCR9 blood cells at day 12, >2,000 cells cultured as 100 cells per well were analyzed.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p

CAF retards melanoma growth in GOF^Notch1 mice.

<p><b>A</b>. Representative images show expression of N1^IC in nuclei (arrowheads pointed) of FSP1⁺ skin fibroblasts of GOF^Notch1 vs. GOF^ctrl mice. <b>B</b>. Expression of transgene-encoded muN1^IC (59 Kd) in skin of GOF^Notch1 mice was detected by Western blotting. β–actin was used as loading control. <b>C</b>. Melanoma growth is retarded in GOF^Notch1 mice (n = 8/group). Five representative images of resected tumors/group are displayed. <b>D</b>. Substantially less Ki67⁺ tumor cells (Luc2⁺) per HPF in melanoma from GOF^Notch1 than GOF^ctrl mice. <b>E</b>. Proliferative activity of melanoma cells (Ki67⁺) is particularly low in the area (marked) adjacent to CAF (FSP1⁺) in GOF^Notch1 mice. Quantifications are counts from 5 HPF per section and 5 section/tumor. The data were statistically analyzed using two-tailed Student’s <i>t</i>-test and expressed as the mean ± SD.</p

CAF promote melanoma invasion in LOF^Notch1 mice.

<p><b>A</b>. <i>Left</i>: Increased melanoma invasion in skin of LOF^Notch1 vs. LOF^ctrl mice (n = 8/group). <i>Right</i>: two representative H&E images of tumor sections per group are displayed. Dash lines indicate tumor boundaries. Arrows point to invading tumor cells. Percentage of invasion is based on results of H&E staining in LPF of each tumor section. <b>B</b>. Null Notch1 CAF which surround tumors exhibit higher proliferative activity as more Ki67⁺/FSP1⁺ cells are detectable in LOF^Notch1 than in LOF^ctrl mice. <b>C</b>. Quantification of Ki67⁺ fibroblasts/HPF in melanoma capsule of LOF^Notch1 (black bar) vs. LOF^ctrl (white bar) mice. Quantifications are counts from 5 HPF per section and 5 section/tumor. The data were statistically analyzed using two-tailed Student’s <i>t</i>-test and expressed as the mean ± SD.</p

CAF inhibit melanoma invasion in GOF^Notch1 mice.

<p><b>A</b>. <i>Left</i>: decreased tumor invasion in GOF^Notch1 compared with GOF^ctrl mice. <i>Right</i>: two representative H&E images of tumor sections (n = 8/group). Dash lines indicate tumor boundaries. Arrows point to invading tumor cells. Percentage of invasion is based on results of H&E staining in low power fields (LPF) of each tumor section. <b>B</b>. Fibroblasts in the melanoma capsule have lower proliferative activity (less Ki67⁺/FSP1⁺ cells) in GOF^Notch1 than in GOF^ctrl mice. <b>C</b>. Quantification of Ki67⁺ fibroblasts/HPF in the melanoma capsule of GOF^Notch1 (black bar) vs. GOF^ctrl (white bar) mice. Results are counts from 5 HPF per section and 5 section/tumor. The data were analyzed using two-tailed Student’s <i>t</i>-test and expressed as the mean ± SD.</p

CAF do not affect melanoma growth in LOF^Notch1 mice.

<p><b>A</b>. Undetectable vs. marginally detectable N1^IC expression in skin fibroblasts of LOF^Notch1 vs. LOF^ctrl mice. Arrowheads point to nuclear staining of N1^IC in fibroblasts. <b>B</b>. Undetectable vs. detectable N1^IC as pointed by arrowheads in fibroblasts at melanoma capsule in LOF^Notch1 vs. LOF^ctrl mice. <b>C</b>. Undetectable full-length of Notch1 protein (271Kd) and slight amount of N1^IC (120 Kd, which was likely presented in non-fibroblasts) in skin tissue of LOF^Notch1 mice was exhibited by Western blotting analysis. β–actin was used as loading control. <b>D</b>. Melanoma growth is comparable in LOF^Notch1 and LOF^ctrl mice (n = 8/group). Five representative images of tumors/group are showed. <b>E</b>. Numbers of Ki67⁺ tumor cells are comparable in LOF^Notch1 (black bar) and LOF^ctrl (white bar) mice. Quantifications are counts from 5 HPF per section and 5 section/tumor. The data were statistically analyzed using two-tailed Student’s <i>t</i>-test and expressed as the mean ± SD.</p

Inhibitory effects of “Notch-activated” fibroblasts as stromal cells on melanoma cell growth and invasion in 3D model.

<p>(<b>A</b>) Representative images of H&E staining of sections of 3D skin melanoma model. (<b>B</b>) Melanoma cell growth in 3D skin model. Significantly decreased melanoma cells were observed in 3D reconstructs in which FF2441-NICD1-GFP cells were embedded in collagen. (<b>C</b>) Decreased invasion into ‘dermis’ by melanoma cells in 3D skin reconstructs in which FF2441-NICD1-GFP cells were embedded. All data are calculated based on that from 5 randomly selected LPF/section and totally 10 sections per group.</p

Notch pathway activation induces expression of WISP-1/CCN4 in fibroblasts.

<p>(<b>A</b>) Levels of mRNA of <i>WISP-1</i>/<i>CCN4</i> were up-regulated in FF2441-NICD1-GFP compared to FF2441-GFP cells. Data are from RT² PCR Array and presented as fold changed in gene expression by setting levels of genes in FF2441-GFP as “1”. Data are presented as mean ± SD of three independently performed experiments. (<b>B</b>) Expression of WISP-1/CCN4 protein was up-regulated by Notch pathway activation in fibroblasts. Expression of WISP-1/CCN4 in FF2441-NICD1-GFP versus FF2441-GFP and untreated F2441 (−) cells was analyzed by Western blotting assay. β-actin was used as loading control. (<b>C</b>) Expression of WISP-1/CCN4 protein was down-regulated in <i>Notch1</i>^−/− MEFs. Expression of WISP-1/CCN4 in <i>Notch1</i>^−/− versus <i>Notch1^Flox/Flox</i> MEFs (both untreated (−) and GFP/lenti-transduced) was analyzed by Western blotting assay. β-actin was used as loading control. (<b>D</b>) Increased WISP-1 promotor-driven luciferase activity in FF2441-NICD1-GFP compared to FF2441-GFP cells. Data are presented as mean ± SD of three independently performed experiments.</p