BRCA1 regulates human mammary stem/progenitor cell fate

Liv et al. 10.1073/pnas.0711613105.

Supporting Information

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SI Figure 7
SI Figure 8
SI Figure 9
SI Table 1
SI Figure 10
SI Table 2

SI Figure 7

Fig. 7. Hierarchy of normal mammary differentiation. (A) Single-cell suspensions of normal mammary cells cultured for 12 days on collagen-coated plates at clonogenic density were sorted by flow cytometry according to the expression of the luminal epithelial marker ESA and the myoepithelial marker CD10. The four cell populations characterized by ESA and CD10 expression were sorted and cultured separately for 12 days on collagen-coated plates. Primitive DN cells (ESA-negative/CD10-negative) give rise to all of the four populations. DP cells (ESA-positve/CD10-positive) are luminal epithelial-committed progenitor cells that give rise to single-positive luminal epithelial cells. Single-positive luminal epithelial cells (ESA-positive/CD10-negative) and myoepithelial cells (ESA-negative/CD10-positive) only give rise to cells with the same phenotype. (B) Model depicting the hierarchy of expression of ESA and CD10 in mammary differentiation.

SI Figure 8

Fig. 8. Effect of BRCA1 knockdown on mammary epithelial cells cultured on collagen plates. Expression of lineage specific markers (CD10 and ESA) was determined by flow cytometry at different time points (0, 7, 12, 26, and 35 days) for BRCA1 siRNA or GFP-control-infected cells cultured on collagen plates. The absolute number of cells for each population and for the two groups is plotted as a function of number of culture days.

SI Figure 9

Fig. 9. Effect of BRCA1 knockdown on mammary epithelial differentiation. Cells derived from BRCA1 siRNA1 lentivirus or GFP-control lentivirus infected cells were induced to differentiate by culturing cells on collagen coated plates and evolution of lineage specific markers (luminal epithelial marker ESA, and myoepithelial marker CD10) was determined by flow cytometry at different time points (0, 7, 12, 26, and 35 days). FACS analysis scatter plots according to ESA and CD10 expression are represented for the two groups for each time point.

SI Figure 10

Fig. 10. Experimental schema for lentivirus infection of mammary epithelial cells. Single mammary epithelial cells isolated from reduction mammoplasties were cultured in suspension in presence of lentivirus (BRCA1 siRNA or GFP-control). Following 1 day of incubation, the viruses were replaced with fresh medium, and cells were cultured in suspension for 1 more day. DsRed or GFP expressing cells were then sorted by flow cytometry and cultured for 7-10 days in suspension to produce mammospheres or on collagen substratum in the presence of FBS to induce differentiation. To evaluate the effect of BRCA1 knockdown on mammary epithelial cells differentiation, evolution of lineage-specific markers was determined at different time point (days 0-35). Time points are noted on experimental schema.

Table 1. BRCA1 mutation status and histoclinical information of 13 BRCA1
carrier patients

Patient no.	BRCA1 mutation type	Cancer development (years pre- and postsurgery)	Age at cancer development	ALDH1 status	Presence of LOH
1	185delAG	Simultaneous	41	Acini Positive	Yes
2	K679X	9 years post	40	Acini Positive	Yes
3	E1353X	Simultaneous	48	Acini Positive	Yes
4	185delAG	Simultaneous	38	Acini Positive	Yes
5	S770X	2 years pre	33	Acini Positive	NA
6	5382insC	3 years free pre and post	NA	negative	No
7	5382insC	Simultaneous	59	negative	No
8	S868X	Pre	NA	negative	No
9	5438insC	Simultaneous	31	negative	NA
10	5382insC	3 years free pre and post	NA	negative	No
11	231delAA	14 years pre	38	negative	NA
12	185delAG	Pre	NA	negative	No
13	IVS17+2T>C	4 years free pre and post	NA	negative	No

NA, none available.

Table 2. Outgrowths produced in humanized fat pad of NOD/SCID mice injected with normal human mammary cells infected with BRCA1 siRNA1 lentivirus or GFP-control lentivirus

Patient no.	No. of fat pads injected (no. of cells injected per fat pad)
Patient no.	Control	BRCA1 siRNA
1	2 (120,000)	2 (120,000)
2	3 (150,000)	3 (150,000)
3	2 (120,000)	2 (120,000)