Fig 2: CDC7 is overexpressed in patient-derived melanoma samples(A) The indicated patient melanoma patient datasets were analyzed for CDC7 mRNA expression using Oncomine, which was upregulated in CDC7 in melanoma samples relative to expression in normal skin.(B) CDC7 mRNA expression was plotted using Gene Expression Profiling Interactive Analysis (GEPIA).(C) CDC7 mRNA expression levels at primary sites and metastasis were plotted for the indicated datasets.(D) Survival analysis (3 years) of patients with melanoma in Human Protein Atlas datasets based on CDC7 expression (low, n = 47; high, n = 55) with p = 0.022.(E) The indicated melanoma patient datasets were analyzed for ELK1 mRNA expression, which was upregulated in melanoma samples relative to normal skin.(F and G) ELK1 mRNA expression levels at primary sites and metastases were plotted for the indicated melanoma datasets using Oncomine.(H) A375 and M14 melanoma cells expressing NS or ELK1 shRNAs were analyzed for ELK1 and CDC7 mRNA expression by RT-qPCR and normalized to ACTB.(I) A375 melanoma cells expressing NS or ELK1 shRNAs were analyzed for ELK1 and CDC7 protein expression by immunoblotting and normalized to ACTINB levels.(J) ELK1 protein enrichment on the CDC7 promoter measured by CUT&RUN assay. ACTB gene promoter regions were used as negative controls. Percent enrichments relative to immunoglobulin G (IgG) under the indicated conditions for each promoter (CDC7 and ACTB) are shown.Data represent the mean ± standard error of three biological replicates. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2.

Fig 3: Effects of BBR on eWAT Gal-3 expression in mice fed a HFD. Male C57BL/6 J mice were grouped and treated as described in “Methods”. At the end of experiment, mice were sacrificed, and eWATs were subjected to paraffin sectioning. After dewaxing, Gal-3 protein expression levels were analyzed by IHC staining(×200, scale bar = 400 µm) (A, upper panel) and integrated optical density (IOD) values (A, lower panel) were determined. Meanwhile, Gal-3 mRNA expression levels in eWATs were analyzed by real-time RT-PCR, normalized to ACTB, and presented as fold of ND group (B). Values are mean ± SD of 10 mice in each group. **p < 0.01, ***p < 0.001 vs. that of ND group; ##p < 0.01, ###p < 0.001 vs. that of HFD group.

Fig 4: Identification of OTUD7A as a Driver Gene of the 15q13.3 Microdeletion(A) Schematic of the human OTUD7A protein showing the exonic 9 bp mutation (de novo p.Asn492_Lys494 deletion in the protein) found in an ASD proband and affected sibling (proband is case 3 in Table 1). The mutation is located in the nuclear localization sequence (NLS) of OTUD7A.(B) OTUD7A mRNA expression relative to ACTB in various human tissues. OTUD7A is enriched in the brain, with highest expression in the frontal cortex.(C) Ratio of brain critical exons in nine genes within the 15q13.3 microdeletion region. FAN1 and OTUD7A contain brain critical exons, with OTUD7A showing a higher ratio than that of FAN1.(D) OTUD7A has the highest pLI (a score that indicates the probability that a gene is intolerant to a loss-of-function mutation) value compared to the other genes in the 15q13.3 critical region. The left y axis represents the number of observed LOF mutations within the ExAC population scale dataset and the right y axis shows the computed pLI score.(E) OTUD7A mRNA expression in the mouse brain is low in early postnatal life, increases into adolescence, and remains stable into adulthood (n = 2 technical replicates).(F) OTUD7A protein co-expression network module. Weighted gene correlation network analysis of OTUD7A from human proteome data shows that 616 genes are highly co-expressed with OTUD7A (top 25th percentile). 21% of genes highly co-expressed with OTUD7A harbor known ASD de novo mutations (left, red dots) and 30% of highly co-expressed genes are targets of FMR1 (right, red dots).(G) DIV14 WT mouse cortical neurons co-expressing Venus and FLAG-OTUD7A WT and co-stained for FLAG and PSD95. OTUD7A is co-localized with PSD95 in dendrites and dendritic spines. Arrows indicate co-localized puncta located in dendritic spines (scale bars = 20 µm, top; 5 µm, bottom).(H) Quantification of FLAG-OTUD7A and PSD95 puncta co-localization showed no changes between OTUD7A WT and OTUD7A p.Asn492_Lys494del overexpression (OTUD7A WT, n = 18 neurons; OTUD7A p.Asn492_Lys494del, n = 15 neurons, 2 cultures, Student’s t test, t(31) = 0.7813). Error bars represent SEM.

Fig 5: LECT2 activates innate immune responses by suppressing Tyr1349 phosphorylation in MET.a HepG2-MET-KO cells were transfected with Control plasmid (Cont), MET WT plasmid (MET WT), or MET mutant plasmid (MET Y1234/1235F, MET Y1349F or MET Y1356F). At 24 h after transfection, the cells were treated with 50 ng/mL r-LECT2 for 1 h and then transfected with HCV-RNA (H77S.3/GLuc-RNA). As a control, HepG2-WT cells were transfected with Cont plasmid; at 24 h after transfection, the Cont plasmid-transfected HepG2-WT cells were treated with 50 ng/mL r-LECT2 for 1 h and then transfected with HCV-RNA. qRT-PCR analysis of IFNB1 in these cells as described above at 12 h after HCV-RNA transfection. Results were normalised to those of ACTB. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001, **p = 0.003, not significant (ns) by two-way ANOVA with Tukey’s multiple comparison test. b HepG2 cells were transfected with Control plasmid (LECT2 (-)) or FLAG-tagged LECT2 plasmid (LECT2 ( + )). At 48 h after transfection, lysates were immunoprecipitated with an anti-MET antibody. Immunoblot analysis of p-MET (Tyr), p-MET (Tyr1234/1235), p-MET (Tyr1349), p-MET (Tyr1356), MET, LECT2 (FLAG) and ß-actin in immunoprecipitated samples and input cell lysate. c HepG2-MET-KO cells were transfected with Control plasmid (EV), MET Y1349D plasmid, or MET WT plasmid. At 24 h after transfection, the cells were treated with 50 ng/mL r-LECT2 for 1 h and then transfected with HCV-RNA (H77S.3/GLuc-RNA). c (left) Immunoblot analysis of p-MET (Tyr1349), MET, RIG-I and ß-actin at 12 h after HCV-RNA transfection. c (right) qRT-PCR analysis of IFNB1 in these cells as described above at 12 h after HCV-RNA transfection. Results were normalised to those of ACTB. Data shown are means of 3 technical replicates ± SEM. ***p < 0.001 by two-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source data file.