Fig 2: AR45 treatment restores the SBMA-associated degeneration in mice.(A) Representative cross sections of gastrocnemius muscle from 11-week-old wild-type and SBMA mice treated with AAV9-control or AAV9-AR45. Sections were stained with H&E and NAD/NADH for muscle morphology. Arrows indicate atrophied myofibers. Arrowheads indicate myofibers containing centralized nuclei. Scale bars, 100 µm. (B) Relative frequency distribution of myofiber cross-sectional diameter size sorted in wild-type, control treated-, and AR45-treated SBMA mice. (C) Representative cross sections of gastrocnemius muscle from 11-week-old SBMA mice treated with AAV9-control or AAV9-AR45 stained with neural cell adhesion molecule (NCAM) antibody, polysialic acid–NCAM (P-NCAM) antibody, and DAPI. Scale bars, 100 µm. (D) Quantification of P-NCAM/NCAM colocalized regions relative to the total number of nuclei is graphed. Quantification was performed from a total of six fields for all groups, three mice per treatment group. (E) Heatmap shows the normalized enrichment scores (NES) of gene sets significantly enriched at P < 0.05 and false discovery rate = 0.25 in the tibilias anterior muscle of AR45-treated compared to control SBMA mice with overexpressed gene sets in red and underexpressed gene sets in blue. Thirty-two pathways were partially restored upon AR45 overexpression. (F) Representative cross sections of lumbar spinal cord and (H) gastrocnemius muscle from 11-week-old wild-type and SBMA mice treated with AAV9-control or AAV9-AR45, stained with 1C2 antibody and hematoxylin. Scale bars, 100 µm. (G and I) Quantification of the number of 1C2-positive staining normalized to the number of cells. Data are means ± SEM. Quantification was performed from a total of six fields for all groups, three mice per treatment group. n.s., not significant.

Fig 3: ICAM1 and NCAM1 are candidate targets of miR-27a in U251 and NPC.a The relative expression of miR-27a transfected by miR-27a mimics and inhibitor in U251 and NPC were determined by qRT-PCR. The two doses in U251 were 30 pmol and 90 pmol and in NPC were 1 pmol and 10 pmol respectively, with five replicates (n = 5) for every sample. b, c Show the relative protein expression levels of NCAM1 and ICAM1 transfected by miR-27a mimics and inhibitor in U251 and NPC, and its quantification using Compass Simple Western software. Date are representatives with three replicates (n = 3) for each sample. Significant differences between these groups were analyzed using one-way ANOVA, Tukey’s multiple comparisons test for adjusted P values. d, e The relative mRNA expression levels of mature miR-27a, NCAM1, and ICAM1 in miR-27 KO using three different sgRNAs compared to WT. Sg30-mix, sg31-mix, and sg32-mix denote the miR-27 KO in U251, n = 5. f The western blot of NCAM1 and ICAM1 in miR-27 KO using three different sgRNAs and WT, n = 3. g The quantification of NCAM1 and ICAM1 protein levels in U251 cells. Significant differences between these groups were analyzed using one-way ANOVA, and Tukey’s multiple comparisons test were used for adjusted P values. Data were shown as mean ± SD. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Fig 4: Clinical validation of tumor-infiltrating immune cells and target genes in clinical osteosarcoma tissues with and without metastasis. (A) Representative images and (B) statistical analyses of immunohistochemical staining for CD56, CD22, CD86, CD163, CD11B, GATA3, LPAR5, EVI2B, RIAM and CFH. Magnification, ×200. *P<0.05.

Fig 5: IL-2 and IL-15 enhance the quantity and cytotoxicity of patient autologous NK cells. Unculture and IL-2 and IL-15 costimulation culture 3 days and 7 days patient autologous CD3-CD56+ NK cell in PBMC detected by FACS (a). The statistical results of CD3-CD56+ NK cell in PBMC (a). Unculture and IL-2 and IL-15 costimulation culture 3 days and 7days CD3-CD56+ NK cell morphology observed under a microscope, respectively (b). The statistical results of CD3-CD56+ NK cell quantity in per microliter (b). Cytotoxic cytokine (CD107a and IFN-R) expression level of NK cells was detected by FACS (c). The proportion of CD107a + IFN-R NK cells (c). For comparisons, Student's t-test was performed in two group, or one-way analysis of variance (ANOVA) was performed in multiple groups. ***p < 0.001 and ****p < 0.0001.