Fig 2: The expression of NGBR in proliferating and involuting phases of infantile hemangioma.(A) The IF staining of PCNA and NGBR increased in proliferating phase hemangioma tissues but decreased in involuting phase hemangioma tissues. Scale bar: 100 µm. (B) The IHC staining of Ki67 increased in proliferating phase hemangioma tissues, and the IHC staining of p16 increased in involuting phase hemangioma tissues. Scale bar: 100 µm. (C) The mRNA levels of NGBR, LYVE1, EGFR, HER2, HER3, CD34, VEGFR2, and FGFR1 were detected by real-time reverse-transcription PCR in hemangioma tissues. *P < 0.05, involuting phase hemangioma tissues (n = 10) vs. proliferating phase hemangioma phase (n = 23). (D) The intense IF staining of NGBR is appreciated in NICH but not in RICH. Scale bar: 100 µm. Statistical analyses: 2-tailed unpaired Student’s t test (C); data are expressed as mean ± SEM. NGBR, NOGOB receptor; IF, immunofluorescence; PCNA, proliferating cell nuclear antigen; NICH, noninvoluting congenital hemangioma; RICH, rapidly involuting congenital hemangioma.

Fig 3: Overactive PI3K signaling links translational dysregulation and aberrant proliferation in FXS-patient-derived cells(A) Representative overlaid dot plots of one control (gray) and one FXS NPC line (red) showing puromycin and Ki67 signals. FXS NPCs have more Ki67+ cells and increased protein synthesis (Mann-Whitney test, *p < 0.05, n = 3 CTR, 3 FXS lines; each data point represents an average of two experimental replicates).(B) Representative images of NPCs stained for Ki67, pHH3 and DAPI after treatment with p110ß inhibitor (TGX) and S6K1 inhibitors (PF). Scale bar, 50 µm.(C and D) Treatment with either inhibitor normalizes aberrant proliferation in FXS NPCs (n = 4 CTR, 4 FXS NPCs; two-way ANOVA, Tukey’s test, *adjusted p < 0.05, **adjusted p < 0.01, ***adjusted p < 0.001, ****adjusted p < 0.0001).(E) Chronic (12-day) treatment using TGX normalizes increased abundance and corrects elevated protein synthesis in proliferating cell populations in FXS cultures. TGX does not significantly alter abundance of non-proliferating cell populations, although they trended toward increased abundance. (n = 4 CTR, 4 FXS NPCs; two-way ANOVA, Tukey’s test, *adjusted p < 0.05, **adjusted p < 0.01, ***adjusted p < 0.001). All data are shown as means ± SEM.

Fig 4: Exosomal RNF157 mRNA suppressed HDAC1 expression to induce the M2 polarization of macrophages. (A) RT-qPCR detected the HDAC1 expression in macrophages after transfection of sh-HDAC1-1/2/3. (B, C) Flow cytometry and RT-qPCR analyzed M2 polarization before and after HDAC1 depletion. (D) The protein levels of RNF157 and HDAC1 were analyzed by western blot in macrophages after co-culturing with PC-3/sh-NC/Exos or PC-3/sh-RNF157-1/Exos. (E) The growth curves of tumors were analyzed. (F) The expression of RNF157 mRNA in tumors was analyzed via RT-qPCR. (G) IHC detected Ki67 level in tumor tissues from different groups. (H) The protein levels of RNF157 and M2 polarization markers (CD163 and ARG1) were analyzed in tumor tissues from different groups. **P < 0.01.

Fig 5: Abnormal proliferation and altered cell-cycle distribution in FXS patient NPCs(A) Representative images of CTR and FXS NPCs stained for Ki67, pHH3, and DAPI. Scale bar, 50 µm.(B and C) High-content image analysis showed FXS patient NPCs have higher mitotic activity as evidenced by more pH3 and Ki67-positive cells normalized to total cells (Mann-Whitney test, **p < 0.01, ***p < 0.001, n = 4 CTR, 4 FXS NPCs, ~3,500 cells imaged per line).(D and E) Colorimetric ELISA shows greater BrdU incorporation in FXS patient NPCs and a FXS isogenic NPC line compared with controls (Mann-Whitney test, **p < 0.01, n = 3 CTR, 3 FXS NPCs, each data point represents an average of two replicates; unpaired t test, *p < 0.05, n = 3 experiments in isogenic control-disease NPC pair. (E) Representative bivariate dot plot for DNA content (FxCycle) and DNA synthesis (EdU). Gates for cells in G0/G1, S, and G2/M phases are indicated.(F) Cell cycle distribution of CTR and FXS NPCs after a 30-min EdU pulse demonstrates significant reduction of cells in G0/G1 and an increase of cells in the S phase. (Two-way ANOVA corrected for multiple comparisons using Holm-Sidak method, *adjusted p < 0.05, **adjusted p < 0.01, n = 3 CTR, 3 FXS NPCs.)(G) Representative images of CTR and FXS 3D organoids stained for proliferative markers Ki67 and SOX2. Scale bar, 50 µm.(H) Quantification of the proportion of Ki67+SOX2+ cells in CTR and FXS organoids at day 28 shows increased actively proliferating cells in FXS (ANOVA, ***p < 0.001, ANOVA, n = 3 CTR, 3 FXS).