Additional file 1 of Inhibition of TGFβ improves hematopoietic stem cell niche and ameliorates cancer-related anemia
Creators: name: Wang, Boyan
name: Wang, Yi
name: Chen, Hainan
name: Yao, Senyu
name: Lai, Xiaofan
name: Qiu, Yuan
name: Cai, Jianye
name: Huang, Yinong
name: Wei, Xiaoyue
name: Guan, Yuanjun
name: Wang, Tao
name: Wang, Jiancheng
name: Xiang, Andy Peng
Publisher: name: figshare
Year: 2021
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Ecology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Fig. S1. General characteristics of LLC mice model. a The tumor volume of tumor-bearing mice since LLC implantation (n=6). b Survival probability of control and LLC-bearing mice over time since LLC implantation (n=20). Data are presented as the means ± SD. Fig. S2. Hematopoiesis was influenced in bone marrow of CRA mice. a Cells number in bone marrow of control and LLC-bearing mice (n=6). b The percentage of myeloid cells in bone marrow of control and LLC-bearing mice (n=6). Data are presented as the means ± SD. *P&lt;0.05, ***P&lt;0.001; assessed by Student’s t test. Fig. S3 LLC-bearing mice exhibited imbalanced bone remodeling. a-c Micro-computed tomography (micro-CT) analysis of trabecular spacing, trabecular pattern factor and trabecular thickness of trabecular bone from the distal femur metaphyses of controls and LLC-bearing mice. Data are presented as the means ± SD of three independent experiments. *P&lt;0.05, **P&lt;0.01; assessed by Student’s t test. Fig. S4 The number of osteoclasts and osteoblasts was increased in cancer cachexia mice. a TRAP stain showing the osteoclasts in trabecular bone from control and LLC-bearing mice. Scale bar, 100 μm. b Immunofluorescence staining of Runx2 showing the osteoblasts in trabecular bone from control and LLC-bearing mice. Scale bar, 100 μm. Fig. S5 Active form of TGFβ1 was increased in CRA mice. a The concentration of active TGFβ1 in the serum of controls and LLC-bearing mice. Data are presented as the means ± SD of three independent experiments. ***P&lt;0.001; assessed by Student’s t test. Fig. S6 SB505124 alleviated the hindered hematopoiesis in LLC-bearing mice. a Experimental design illustrating subcutaneous injection of DMSO or SB505124 (5 mg/day/kg) into control and LLC-bearing mice (n=6/group). b-e The percentage and number of Ter119+ cells in cluster III and cluster V in bone marrow of control, LLC and LLC+SB505124 mice. f The number of white blood cells in peripheral blood of control, LLC and LLC+SB505124 mice. g The percentage of myeloid cells in bone marrow of control, LLC and LLC+SB505124 mice. Data are presented as the means ± SD of three independent experiments. *P&lt;0.05, **P&lt;0.01, ***P &lt; 0.001; assessed by Student’s t test. Fig. S7 SB505124 inhibited TGFβ signaling activation in bone marrow and MSC. a-b Quantifications of the Western blotting of phosphorylated Smad2/3 and total Smad2/3 proteins in bone marrow of control, LLC and LLC+SB505124 mice (n=6/group). c mRNA expression of TGFβ target genes in MSC of control, LLC and LLC+SB505124 mice (n=6/group). Data are presented as the means ± SD of three independent experiments. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001; assessed by Student’s t test. Fig. S8 SB505124 relieved the osteolytic process of LLC-bearing mice. a The mRNA levels of osteoclastic genes in bone marrow of control, LLC and LLC+SB505124 mice (n=6/group). b Representative three-dimensional thickness maps from micro-CT scans of trabecular bone from the distal femur metaphysis of control, LLC and LLC+SB505124 mice. Scale bar, 1000 μm. c-f Ratio of bone surface area to bone volume, ratio of bone volume to total volume, trabecular number, and cortical wall thickness of control, LLC and LLC+SB505124 mice (n=3/group). Data are presented as the means ± SD of three independent experiments. *P&lt;0.05; assessed by Student’s t test. Supplemental Table S1. Primer used to amplify the mouse transcripts during PCR. Supplemental Table S2. Primary and secondary antibodies.