Supplementary figure1
LRRC16B expression in the embryonic and adult rats.SD rats from the Animal Institute of National Cheng Kung University (NCKU) were handled based on guidelines approved by the Institutional Animal Care and Use Committee. The day of plug formation of female rat was considered as E0.5.(A) Expression pattern of rat-GDF11, rat-IMP-1, rat-FGF-18, and rat-LRRC16B was examined by RT-PCR analysis of cDNA from different embryonic periods (E8.5 to E17.5), and in the brain, liver, lung, heart, spleen and kidney on post-natal day 0 (P0) rats. Rat-GDF11 was initially expressed in E11.5 and persisted to P0. Rat-IMP-1 was initially expressed in E11.5, peaked in E14.5, and presented in most organs at P0. Rat-FGF18 was initially expressed in E8.5, peaked in E14.5, but disappeared in E17.5 and P0. Rat-LRRC16B was detected in E11.5, E14.5, and E17.5; and in the brains, lungs, hearts, and kidneys of P0 rats. (B)The brain, liver, lung, heart, stomach, uterus, urinary bladder, spleen, and kidney were dissected from adult rats aged 15 weeks.In adult rats,rat-GDF11, rat-IMP-1, rat-FGF18, and rat-LRRC16B were not detected, except for rat-LRRC16B, which was expressed in the brain.
Supplementary figure2
Sub-cellular localization of theLRRC16B-EGFP fusion protein in the 293T stable
pool cells. Representative images of 293T cells were expressing EGFP orLRRC16B-EGFP fusion proteins. Bar was 10μm.
Supplementary figure3
Validation of the knockdown effect of RNAi constructs. The transfections were performed in a 6-well plate using 2 × 105cells per well for 293T in 2 ml DMEM with 10% FBS 100Uml-1 penicillin, and 100 mg ml-1 streptomycin per well. For each well, co-transfection 1 μg LRRC16B-RNAi-1~4 and 1 μg LRRC16B-EGFP and 2 μl Lipofectamine™ 2000 were used. The construct and the Lipofectamine™ 2000 were incubated together for 20 min in 200 μl serum-free DMEM, and then added to the cell suspensions that included 800μl serum-free DMEM. These were incubated at 37°C for 6 h before extra 2ml DMEM with 10% FBS 100Uml-1 penicillin and 100 mg ml-1 streptomycin were added. After 48 hours, image analysis(A) and western blot (B)against EGFP antibody were performed to evaluate theRNAi knockdown efficiency in 293T cell lines. Bar was 50μm.
Supplementary figure4
Immunohistochemistry of human fetal tissues. A fetal tissue array was purchased from Biomax (US Biomax;Rockville, MD, USA).The immunostaining was performed according to the manufacture’s instruction. The tissue array was incubated with apolyclonal anti–LRRC16B antibody (HPA030596 from Sigma, St. Louis, MOUSA)with a dilution of 1:40 at 4℃overnight. The EnVision®+ System-HRP (DAB) kit(Dako Denmark; Glostrup, Denmark) was used to detect the resulting immunecomplex. Peroxidase activity was visualized by3,3'-diaminobenzidine (DAB). Finally, the tissue array was counterstained with hematoxylin.The antibody highlighted the neurons but not glial cells in the human fetal brain (A). There were signals in the epithelial cells of fetal colon, kidney, and lung (B, C, and D). Immunohistochemical signal was also noted in the mesenchymal cells in the lamina propria of fetal colonic mucosa (B).