Fig 2: miR-29c-3p targets NSD1 mRNA in differentiated HFSCs. (A) Detection of miR-29c-3p binding to NSD1 mRNA using the luciferase activity reporter assay. *p < 0.05 compared with treatment of mimic NC. (B) NSD1 mRNA expression after differentiation of HFSCs into sebaceous gland cells as determined by RT-qPCR. *p < 0.05 compared with HFSCs. (C) NSD1 protein level after differentiation of HFSCs into sebaceous gland cells as determined by Western blot analysis. *p < 0.05 compared with HFSCs. (D) NSD1 fluorescence intensity after differentiation of HFSCs into sebaceous gland cells as determined by immunofluorescence assay (scale bar = 25 μm). *p < 0.05 compared with HFSCs. (E) NSD1 mRNA expression after differentiation of HFSCs into epidermal cells as determined by RT-qPCR. *p < 0.05 compared with HFSCs. (F) NSD1 protein level after differentiation of HFSCs into epidermal cells as determined by Western blot analysis. *p < 0.05 compared with HFSCs. (G) NSD1 fluorescence intensity after differentiation of HFSCs into epidermal cells as determined by immunofluorescence assay (scale bar = 25 μm). *p < 0.05 compared with HFSCs. (H) miR-29c-3p expression levels in HFSCs treated with miR-29c-3p mimic as determined by RT-qPCR. (I) NSD1 mRNA expression in HFSCs treated with miR-29c-3p mimic as determined by RT-qPCR. *p < 0.05 compared with treatment of mimic NC. (J) NSD1 protein level in HFSCs treated with miR-29c-3p mimic as determined by Western blot analysis. *p < 0.05 compared with treatment of mimic NC. (K) miR-29c-3p expression levels in HFSCs treated with miR-29c-3p inhibitor (plasmid suppressing the expression of miR-29c-3p) as determined by RT-qPCR. (L) NSD1 mRNA protein expression in HFSCs treated with miR-29c-3p inhibitor as determined by RT-qPCR. *p < 0.05 compared with treatment of mimic NC. (M) NSD1 protein level in HFSCs treated with miR-29c-3p inhibitor as determined by Western blot analysis. *p < 0.05 compared with treatment of mimic NC. The data are expressed as mean ± standard deviation, and comparisons between two groups were analyzed by unpaired t test. The experiment was repeated three times.

Fig 3: DLX5 induces HFSC differentiation by regulating the c-MYC/miR-29c-3p/NSD1 axis. (A) DLX5, NSD1, and c-MYC mRNA expressions in HFSCs as determined by RT-qPCR. (B) DLX5, NSD1, and c-MYC protein levels in HFSCs as determined by Western blot analysis. (C) miR-29c-3p expression levels in HFSCs were measured using RT-qPCR. (D) CCK-8 assay used to detect the proliferation rates in HFSCs. (E) Oil red O staining before and after differentiation of HFSCs into sebaceous gland cells (scale bar = 25 µm). (F) Detection of EMA and CD200 expression before and after differentiation of HFSCs into sebaceous gland cells using flow cytometry. (G) Detection of keratin CK10 and CD200 expression before and after the differentiation of HFSCs into epidermal cells using flow cytometry. *p < 0.05 compared with treatment of oe-NC + sh-NC; #p < 0.05 compared with treatment of oe-DLX5 + sh-NC. The data were expressed as mean ± standard deviation. Comparisons among multiple groups were performed using one-way ANOVA and Tukey’s test. Comparisons among multiple groups at different time points were performed using two-way ANOVA and Bonferroni test. The experiment was repeated three times.

Fig 4: KB-68A7.1 bound to NSD1 and redistributed NSD1 to the cytoplasm. (A) Subcellular distribution of KB-68A7.1 in SNU-398 cells was assessed by RNA FISH. (B) Subcellular distribution of KB-68A7.1 in SNU-398 cells was assessed by cytoplasmic and nuclear RNA purification, followed by RT-qPCR. GAPDH and MALAT1 were used as cytoplasmic and nuclear RNA controls, respectively. (C) RIP assays were undertaken in SNU-398 cells using NSD1 primary antibody or non-specific IgG. Enrichment of RNA was assessed by RT-qPCR. (D) RNA–protein pull-down assay were undertaken in SNU-398 cells using RNA end-labeled with desthiobiotin. Enrichment of protein was assessed by Western blot. (E) Nuclear and cytoplasmic NSD1 levels in SNU-398 cells with KB-68A7.1 overexpression or control was measured by Western blot. Histone H3 and GAPDH were used as loading controls for nuclear and cytoplasmic proteins, respectively. (F) Nuclear and cytoplasmic NSD1 levels in SNU-398 cells with KB-68A7.1 silencing or control was measured by Western blot. (G) Subcellular distribution of NSD1 in SNU-398 cells with KB-68A7.1 overexpression or control was assessed by IF. (H) Subcellular distribution of NSD1 in SNU-398 cells with KB-68A7.1 silencing or control was assessed by IF. Results are shown as mean ± SD based on three independent experiments. **p < 0.01, ns, not significant by two-tailed unpaired t-test.

Fig 5: NSD1 overexpression reverses the effects of miR-29c-3p to promote HFSC differentiation. (A) miR-29c-3p expression in HFSCs was determined by RT-qPCR. (B) NSD1 mRNA expression in HFSCs determined by RT-qPCR. (C). NSD1 protein level in HFSCs determined by Western blot analysis. (D) CCK-8 assay used to detect the proliferation rate of HFSCs. (E) Oil red O staining before and after differentiation of HFSCs into sebaceous gland cells. (F) Detection of EMA and CD200 expression before and after the differentiation of HFSCs into sebaceous gland cells using flow cytometry. (G) The detection of keratin CK10 and CD200 expression before and after the differentiation of HFSCs into epidermal cells using flow cytometry. *p < 0.05 compared with treatment of mimic NC + oe-NC. #p < 0.05 compared with treatment of miR-29c-3p mimic + oe-NC. The data are expressed as mean ± standard deviation. Comparisons among multiple groups were performed using one-way ANOVA and Tukey’s test. Comparisons among multiple groups at different time points were performed using two-way ANOVA and Bonferroni test. The experiment was repeated three times.