Additional file 1 of Repopulating Kupffer cells originate directly from hematopoietic stem cells

Additional file 1. Fig S1. Intraperitoneal injection with 20mg/kg clodronate-liposomes did not deplete bone marrow macrophages and did not trigger liver inflammation. A Flow cytometric analysis of bone marrow-derived mononuclear phagocytic system (BM MPS) cells of C57/BL mice received indicated dose of clodronate-liposomes injection (n = 5/group). B Cell count of BM MPS of C57/BL mice treated with the indicated dose of clodronate-liposomes analyzed in A. C Liver tissue from all normal-saline treatment mice at each time point revealed normal cellular architecture (n = 5). Liver tissue from the clodronate-liposomes group revealed no damage to liver cells and inflammatory cells infiltration (n = 5). Liver tissue from the carbon tetrachloride treatment group revealed some damage to liver cells, inflammatory cells infiltration, fatty changes, and centrilobular necrosis (n = 5), scale bar = 50 μm. D Serum alanine aminotransferase of mice from the carbon tetrachloride group was significantly increased at 24 hours posttreatment, and returned to normal level at 96 hours (n = 5/group). In contrast, serum alanine aminotransferase of mice from the clodronate-liposomes group and normal saline group was remained unchanged, at the meantime. Fig S2. Analysis of kupffer cells (KCs) from C57BL/6 mice following intraperitoneal injection with 20mg/kg clodronate-liposomes. A GFP expression on CD68+ and CD68− KCs from E8.5 pulsed Cre mice at 8 weeks after birth. B Flow-cytometric analysis of KCs from C57BL/6 mice 24 hours after being treated with intraperitoneal injection of clodronate-liposomes of indicated dose (n = 6/group). C Percentage of KCs from C57BL/6 mice treated with intraperitoneal injection of clodronate-liposomes of indicated dose analyzed in B. Fig S3. Proliferating ration of bone-marrow-cells (BMCs) and labeled/unlabeled kupffer cells (KCs) from E8.5-pulsed Csf1RCreERT2; RosamT/mG mice at 10 day and 90 day post-intraperitoneal injection with 20mg/kg of control-liposomes. A Representative results of percentage of EdU+ BMCs and labeled/unlabeled KCs. B Percentage of EdU+ BMCs or labeled/unlabeled KCs from indicated mice analyzed in A. Values are the means ± SEM from 6 samples. ***P < 0.001, N.S No significant difference between each Edu/no-Edu group by t-test. Fig S4. Flow cytometric analysis of GFP expression of hematopoietic stem cells and blood leukocytes within purified GFP+ HSC-chimeric Kitw/Kitwv mice. Fig S5. Flow cytometric analysis of YFP expression of indicated cells within adult pulsed Cx3cr1CreERT2; RosaYFP mice (Cre) or Cx3cr1wt; RosaYFP mice (No cre). (A) Flow-cytometric analysis of bone marrow monocytic cells and blood MO from adult pulsed Cx3cr1wt; RosaYFP or Cx3cr1CreERT2; RosaYFP mice at indicated time point post pulse (n = 5/group). (B) Gating strategy of intra-splenic MO. Dot plots are gated on viable single splenic cells. Intra-splenic MO are defined as Ly6C+ cells. (C) Flow cytometric analysis of YFP expression on intra-splenic MO and KCs within the same adult pulsed Csf1rMeriCreMer; RosaYFP mice at indicated time point post intraperitoneal injection of 20mg/kg Clo (n = 4/group). (D) Flow-cytometric analysis of blood leukocytes and KCs from adult pulsed Csf1rMeriCreMer; RosaYFP mice at 25-day post pulse (n = 5/group). (E) Flow-cytometric analysis of blood leukocytes and KCs from adult pulsed Csf1rwt; RosaYFP mice at 25-days post pulse (n = 5/group). (F), (G), (H), (I) Labeling index of intra-splenic MO and KCs at indicated time point post intraperitoneal injection of 20mg/kg Clo, analyzed in (C). *** P < 0.001. Fig S6. Flow-cytometric analysis of bone-marrow hematopoietic stem cells from C57BL/6 mice at indicated time point post intraperitoneal injection with 20gm/kg clodronate-liposomes (n = 5/group). Fig S7. Flow-cytometric analysis of blood hematopoietic stem cells (HSCs) from C57BL/6 mice at indicated time point post intraperitoneal injection with 20mg/kg Clo (n = 5/group). HSCs were defined as LinnegSca-1+c-kit+CD34+CD135− cells. Fig S8. Flow cytometric analysis of GFP liver non-parenchymal cells in kupffer cells-depleted mice revived GFP+ hematopoietic stem cells engraftment. Fig S9. Percent of GFP kupffer cells (KCs) from KC-depleted and purified GFP+ hematopoietic stem cellsengrafted, KC-depleted and purified GFP multipotent progenitors engrafted, or KC-depleted and purified GFP+ monocytic cells engrafted C57BL/6 mice at 90-day post-engraftment. Values are the means ± SEM from 6 samples. ***P < 0.001 between groups by ANOVA. Fig S10. Gating strategies of kupffer cells (KCs), bone marrow-derived mononuclear phagocytic system cells, macrophages, and blood leukocytes. A Gating strategy of non-parenchymal liver cells (NPCs). Dot plots are gated on total liver NPCs, 7-AAD+ dead cells, and doublets were excluded from the analysis and sorting. B Gating strategy of KCs. Dot plots are gated on viable single liver NPCs. KCs are defined as F4/80+, CD45+ C, CD11b+ D, and Ly6C− E cells. F Gating strategy of bone marrow hematopoietic stem cells. 7-AAD+ dead cells and doublets were excluded from the analysis and sorting. Dot plots are gated on all bone marrow cells, then on Lineage-cells, then on Sca-1+ and c-kit+ cells, and finally on CD34− CD135− cells. Hematopoietic stem cells are defined as CD34− and CD135− CD48− CD150+ LSK cells. LT-HSC is defined CD34− and CD135− CD48− CD150− LSK cells, ST-HSC is defined as CD34+ and CD135− CD48− CD150+ LSK cells, MMP2 is defined as CD135− CD34− CD150+ CD48+ LSK cells, MMP3 is defined as CD135− CD34+ CD150− CD48−, MMP4 is defined as CD135+ CD34+ CD150− CD48+ LSK cells. G Gating strategy of bone marrow monocytic progenitors. H Gating strategy of bone marrow macrophages cells. I Gating strategy of blood B cells. J Gating strategy of blood T cells. K Gating strategy of blood monocytic cells. Dot plots are gated on blood cells in the monocyte region. Monocytic cells is defined as CD115+ cells. M Gating strategy of blood neutrophilia granulocyte