Fig 2: Cell proliferation activity assays of PDCD4 knockdown LX-2 cells. Control cells on day 1 and negative control and PDCD4-specific (p2 and k603) siRNA-treated LX-2 cells on days 1, 2, and 3 were stained with anti-Ki-67 antibody and 7-AAD and analyzed by flow cytometry. After the exclusion of dead and doublet cells, single cells were gated in a combination of Ki-67 and 7-AAD staining. This experiment was repeated four times independently, and the upper panel represents the gate of one experiment. The diagram shows the average percentage of Ki-67-positive cells of the four experiments (lower panel). t test, *p < 0.05, **p < 0.005

Fig 3: Lipolysis is required for CD81+ APC proliferation(A) Schematic illustration of the experiments.(B) Relative mRNA levels of Pnpla2 in indicated tissues. n = 4.(C) Representative H&E staining and immunofluorescent staining for UCP1 (red)/DAPI (blue) in the inguinal WAT of mice. LN, lymph node. Scale bars, 100 µm.(D) Relative mRNA levels of indicated genes in the inguinal WAT. n = 6 for Adipo-ATGL KO mice, n = 4 for controls.(E) Quantification of Ki67+ CD81+ cells in the inguinal WAT. n = 5 at 30°C, n=7 at 8°C.(F) Quantification of Ki67+ CD81+ cells in ex vivo cultured inguinal WAT. Adipose tissues were treated with vehicle or CL316,243 at 1 µM. n = 3.(G) Quantification of Ki67+ CD81+ cells in ex vivo cultured inguinal WAT that were treated with atglistatin at 50 µM and/or CL316,243 at 1 µM. n = 4 for vehicle, and n = 5 for CL316,243.(B and D) *p < 0.05, **p < 0.01, ***p < 0.001, by two-tailed unpaired Student’s t test. (E–G) *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA followed by the Tukey-Kramer’s post hoc test.

Fig 4: Cold and ß3-AR stimuli induce CD81+ APC proliferation(A) Top: schematic illustration. Male mice at 10 weeks old were treated with tamoxifen at 30°C and exposed to 8°C for 3 days. Bottom: relative mRNA levels of Pparg in isolated CD81+ cells. n = 4.(B) Representative H&E staining of the inguinal WAT in (A). LN, lymph node. Scale bars, 100 µm.(C) Relative mRNA levels of indicated genes in the inguinal WAT following cold exposure. n = 6 for PpargCD81 KO, n = 4 controls.(D) Quantification of Ki67+ CD81+ cells in the inguinal WAT of mice at 30°C and 8°C for 1 day and 4 days. n = 5 for 30°C and 8°C for 1 day, n = 8 for 8°C for 4 days.(E) Left: representative immunofluorescent staining for GFP (Green)/Ki67 (Red) in the inguinal WAT of Cd81-lineage reporter mice following cold exposure. Scale bars, 100 µm. Right: magnified images. The arrowheads show GFP+ Ki67+ cells. Scale bars, 50 µm.(F) Quantification of GFP+ Ki67+ cells in (E). n = 4.(G) Quantification of Ki67+ CD81+ cells in the inguinal WAT of mice. Left: mice kept at 30°C or 8°C for 3 days. Right: mice treated with vehicle or CL316,243 for 3 days. n = 4.(H) Schematic illustration of ex vivo studies. Mouse inguinal WAT or human subcutaneous WAT were cultured with CL316,243 (0.1–1 µM in mice, 1 µM in human sample). Ki67+ CD81+ cells in the WAT samples were quantified by FACS.(I) Quantification of mouse Ki67+ CD81+ cells in (H). n = 3.(J) Quantification of human Ki67+ CD81+ cells in (H). n = 3 technical replicates for each biopsied sample (7 pairs). p value was determined by multilevel analysis. (A, C, F, and G) *p < 0.05, **p < 0.01, by two-tailed unpaired Student’s t test. (D and I) *p < 0.05, ****p < 0.0001, by one-way ANOVA followed by the Tukey-Kramer’s post hoc test. ns, not significant.

Fig 5: Linoleic acid stimulates CD81+ APC proliferation(A) Schematic illustration of the experiments. See text for details.(B) Relative cell number (normalized to each vehicle) of inguinal WAT-derived CD81+ cells in (A). n = 4.(C) Candidates of fatty acids studied in the study.(D) Heatmap of cell numbers in CD81+ cells treated with indicated fatty acids for 4 days. The color scale shows Z score levels of the relative cell number. n = 4. p value by two-way repeated-measures ANOVA is shown in each panel.(E) Quantification of CD81+ cells in indicated cell cycle phases. n = 3.(F) The cell number of inguinal WAT-derived CD81+ cells treated with indicated molecules for 4 days. n = 4. ***p < 0.001 by one-way ANOVA followed by the Tukey-Kramer’s post hoc test.(G) Schematic illustration of linoleic acid supplementation.(H) Quantification of Ki67+ CD81+ cells in the inguinal WAT following linoleic acid supplementation at 30°C. n = 7.(I) Relative mRNA levels of indicated genes in the inguinal WAT following cold exposure. n = 9.(J) Representative immunofluorescent staining for GFP (green)/UCP1 (red) in the inguinal WAT of Cd81-lineage reporter mice following linoleic acid supplementation and cold exposure. Scale bars, 100 µm.(K) Relative mRNA levels of indicated genes in the inguinal WAT following linoleic acid supplementation and cold exposure. n = 6.(L) Representative H&E staining in the inguinal WAT. LN, lymph node. Scale bars, 100 µm.(B,E, H, I, and K) *p < 0.05, **p < 0.01, ***p < 0.001, by two-tailed unpaired Student’s t test. ns, not significant.