Loss of Ptn affects hematopoiesis in vitro. (A) Expression of Ptn in UG26-1B6 and EL08-1D2 cell lines compared with a mixture of nonsupportive stromal cell lines (UG15-1B7, EL28-1B3, and AM30-3F4) as detected by real-time PCR. Shown is expression relative to housekeeping gene Ythdf1. (B) Knockdown of Ptn in supportive stromal cell lines as measured by real-time PCR. Shown is expression relative to Ythdf1. Gray bars represent the unmanipulated parental stromal cells, white bars represent the stromal cells transduced with empty pLKO.1 vector (pLKO.1), and shPtn-transduced cells (Ptn^KD) are shown in black bars. (C) Western blot analysis of Ptn expression, using supernatant of confluent monolayer of stromal cell lines. (D) Colony number of WT Lin⁻ after 2 weeks of coculture on Ptn^KD and pLKO.1 stromal cell lines (n = 10, (UG26-1B6 contact; n = 4, UG26-1B6 noncontact; n = 3, EL08-1D2 contact). (E) The colony number of Ptn^+/+ and Ptn^−/− Lin⁻ cells after 2 week of coculture on Ptn^KD and pLKO.1 UG26-1B6 (n = 4). All values are mean ± SEM; *P < .05.