Figure S2.

Autocrine effect of MPP3 secretion. (A–D) Secretory activity of HSPCs at the single cell level with (A) experimental scheme of HSC, MPP3, and MPP4 single-cell secretion assay ± LPS/Pam3CSK4 (L/P) stimulation for 18 h in culture (14 known cytokines were preselected for this assay); (B) heatmap of secretion frequency by single unstimulated/stimulated HSC, MPP3, and MPP4; (C) TNFα and IL-6 secretion intensity by all unstimulated/stimulated secreting MPP3. Cells with fluorescence (Fluo.) signal intensity above threshold (set to 2) are counted as secretors, and black lines represent mean values; and (D) TNFα and IL-6 secretion frequency and secretion intensity by IL-10–secreting MPP3. Data are from four independent experiments. (E) Model depicting the effect of IL-10 on TNFα and IL-6 secretion by individual MPP3. (F) Changes in IL-6 secretion by MPP3 upon IL-10 addition (10, 30, or 100 ng/ml), TNFα addition (1 µg/ml), TNFα genetic deletion, and NF-κB (BMS345541, 2 µM) or Ca²⁺ (KN-93, 2 µM) signaling inhibition. Supernatants were collected upon the culture of 10,000 WT or Tnf^−/− MPP3 for 24 h in 150 µl base media or full cytokine media (for TNFα addition) ± LPS/Pam3CSK4 (L/P) stimulation and the indicated inhibitor. Results are ELISA measurements (two independent experiments). (G) Differential secretion of MIP1α and GM-CSF by ER^high vs. ER^low MPP3 upon stimulation. Results are Luminex cytokine bead array measurement of 24-h supernatants (two independent experiments). Data are means ± SD except when indicated, and significance was assessed by a two-tailed unpaired Student’s t test.