Supplementary Figures

Transcription

1 Supplementary Figures Fig. S1. Specificity of perilipin antibody in SAT compared to various organs. (A) Images of SAT at E18.5 stained with perilipin and CD31 antibody. Scale bars: 100 μm. SAT stained with guinea pig IgG as a negative control (top) or guinea pig antimouse perilipin antibody (bottom), followed by Cy3-conjugated goat-origin anti-guinea pig antibody. (B) Images of indicated organs at E18.5 stained with perilipin and CD31 antibody as a negative control of perilipin staining compared to SAT. Scale bars: 100 μm.

2 Fig. S2. Identification of proliferative cells in the clusters. (A) Image showing the border of inguinal WAT at E18.5. Scale bar: 100 μm. Yellowishred dashed line indicates vascular margin, whereas white dashed line indicates adipose margin. (B) Image of whole inguinal WAT stained for perilipin and PH3. Scale bar: 500 μm. (C) Image of whole inguinal WAT stained for perilipin, PPARγ and EdU. EdU was injected into the peritoneal cavity of pregnant female 3 hr before harvest. Scale bar: 250 μm. (D) Images showing 3T3-L1 cells at day 3 after adipogenic differentiation. Arrows indicate perilipin + PPARγ + PH3 + cells, and arrowhead indicates perilipin - PPARγ + PH3 + cell. (E) Serial images taken with a live imaging device at 10 min intervals. BODIPY stained (arrow) 3T3-L1 cells at day 2 after adipogenic differentiation are divided into two daughter cells (arrowhead). Scale bars, 10 μm.

3 Fig. S3. Recombination efficiency of Pdgfrb-Cre-ER T2 neonatal mice in adipose tissues and retina. Pdgfrb-Cre-ER T2 /tdtomato mice were analyzed to examine Cre-mediated recombination efficiency. (A) Schematic diagram of the Pdgfrb-Cre-ER T2 transgene and R26-tdTomato construct, and tamoxifen administrations (Cre induction, red

4 arrowheads) schedule. Neonatal mice received two injections of tamoxifen with a twoday interval after birth, and were analyzed one week after last injection. (B) Inguinal WAT (top), BAT (middle), and retina (bottom) were immunostained with antibodies against CD31 and PDGFRβ, and compared with tdtomato signals. Scale bars: 50 μm. (C) Relative percentage of PDGFRβ immunostained cells that are labelled with tdtomato signals. Almost 100% of the recombination efficiency is shown. Three independent experiments showed similar results.

5 Fig. S4. Differentiation potential of PPA in ex vivo culture. (A,B) Clusters derived from E18.5 were harvested and cultured ex vivo for 7 days with culture medium (control) or adipogenic differentiation medium. Asterisk indicates cluster. (A) Photographs of clusters and proliferated cells around cluster stained with Oil Red O. Arrows indicate newly formed adipocytes from the proliferated cells. (B) Images of cluster and proliferated cells around cluster under the control culture medium. The region outlined by a dotted square is magnified (right panel). Scale bars: 50 μm.

6 Fig. S5. Prenatal adiponectin + SVF cells include functional preadipocytes. The SVF cells derived from E18.5 SAT of Adiponectin-Cre/tdTomato embryos were sorted into tdtomato - cells and tdtomato + cells, and cultured under the adipogenic differentiation medium for 5 days. (A,B) Images of tdtomato + SVF cells stained with various antibodies. (A) Arrows indicate tdtomato + SVF cells containing numerous perilipin + lipid droplets. Scale bars: 20 μm. (B) Arrow indicates tdtomato + SVF cell expressing PH3, proliferation marker. Scale bars, 100 μm. (C) Images of tdtomato - and tdtomato + SVF cells during adipogenic differentiation. (D) Photographs of Oil Red O staining of tdtomato - and tdtomato + SVF cells 5 days after adipogenic differentiation.

7 Fig. S6. FACS gating strategy for isolation of perilpin + or adiponectin + cells among lineage-negative population. Representative dot plots showing FACS staining profiles and gating of SVF cells from P0 SAT. (A) Singlet population was selected by exclusion of doublets and clumps by gating. Lineage-negative cells were chosen for lack of CD31, CD45, and Ter119 expression. (B) Unstained control (left), fluorescence minus one (FMO) control (middle), or fully stained sample with perilipin antibody (right).

8 Fig. S7. Flow cytrometry plots of PPAs co-expressing preadipocyte markers. (A) Flow cytometry plots of perilipin + SVF cells with surface markers such as CD24, CD29, CD34, Sca-1 and PDGFRα in flow cytometry analyses among lineagenegative population at E18.5. (B) Relative proportion of cells with CD24, CD29, CD34, Sca-1 and PDGFRα among perilipin + SVF cells at E18.5. n = 4 per group. Values are mean ± SD. The p values were determined by the Kruskal-Wallis test followed by Tukey post hoc test. * p < 0.05 versus CD24; # p < 0.05 versus CD29; $ p < 0.05 versus CD34 or Sca-1. Error bars represent s.d.

9 Supplementary Table Table S1. Antibodies used. Antibody Clone Manufacturer Immunostaining Hamster anti-mouse CD31 2H8 Millipore Rabbit anti-mouse C/EBPα Polyclonal Cell Signaling Rat anti-mouse Integrin α5 5H10-27 BD Pharmingen Rabbit anti-mouse laminin Polyclonal Abcam Guinea pig anti-mouse perilipin Polyclonal Fitzgerald Rat anti-mouse PDGFRβ APB5 Abcam Rabbit anti-mouse PH3 Polyclonal Millipore Rat anti-mouse PH3 HTA28 Abcam Rabbit anti-mouse PPARγ 81B8 Cell Signaling Flow cytometry Rat anti-mouse CD16/32 93 BioLegend APC-conjugated anti-mouse CD24 M1/69 BD Pharmingen APC-conjugated anti-mouse CD29 HMβ1-1 AbD Serotec APC-conjugated anti-mouse CD34 MEC14.7 BioLegend APC-conjugated anti-mouse Sca-1 D7 ebiosciences APC-conjugated anti-mouse CD140a APA5 ebiosciences FITC-conjugated anti-mouse CD ebiosciences FITC-conjugated anti-mouse CD45 30-F11 ebiosciences FITC-conjugated anti-mouse Ter119 Ter-119 ebiosciences Guinea pig anti-mouse perilipin Polyclonal Fitzgerald

10 Table S2. Secondary Antibodies used. Antibody Host Cat no. Manufacturer Cy3-conjugated antiguinea pig Cy5-conjugated antiguinea pig FITC-conjugated antiguinea pig Cy3-conjugated antihamster Cy5-conjugated antihamster Goat Goat Goat Goat Goat Cy3-conjugated anti-rabbit Goat Cy5-conjugated anti-rabbit Goat FITC-conjugated anti-rabbit Goat Cy3-conjugated anti-rat Goat FITC-conjugated anti-rat Goat Cy3-conjugated anti-goat Donkey FITC-conjugated anti-goat Donkey

11 Table S3. Primers used for quantitative real-time PCR. Gene Sequences (5-3 ) Adiponectin Cebpa Cebpb Ki67 Plin1 Pparg2 tdtomato Vegfr2 Forward 5 -ACATCCTGGCCACAATGGCACAC-3 Reverse 5 -GTCTCACCCTTAGGACCAAGAAG-3 Forward 5 -GACAGAGACCTAGGTTTCTGGGCTTT-3 Reverse 5 -GGACACAGAGACCAGATACAAGTGTTG-3 Forward 5 -AAGCTGAGCGACGAGTACAAGATG-3 Reverse 5 -TTGAACAAGTTCCGCAGGGTGCT-3 Forward 5 -ATTCGTATCCAGCTGCCTGTAGTGTC-3 Reverse 5 -GGCTTGCTTCCATCCTCATGATTTCC-3 Forward 5 -GTGCAATGCCTATGAGAAGGGTGTAC-3 Reverse 5 -GTAGAGATGGTGCCCTTCAGTTCAGA-3 Forward 5 -TGCACTGCCTATGAGCACTTCACA-3 Reverse 5 -AGGAATGCGAGTGGTCTTCCATCA-3 Forward 5 -TACTACTACGTGGACACCAAGCTG-3 Reverse 5 -ATGACGGCCATGTTGTTGTCCTCG-3 Forward 5 -TATTGGTGACTGAATGCGGCGGTG-3 Reverse 5 -ATGTACACGATGCCATGCTGGTCAC-3