Fig 2: S100A6 is directly transcriptionally regulated by c-Myc (A, B) The mRNA (A) and protein (B) expressions of c-Myc and S100A6 were determined in HaCaT cells with c-Myc overexpression or knockdown. (C, D) The protein expression levels of c-Myc and S100A6 were determined in HaCaT cells pre-treated with 10058-F4 (20 μM). (E) The predicted binding site of c-Myc in the S100A6 promoter region on the Jaspar database (http://jaspar.genereg.net/)(relative profile score threshold 90%) and scheme of mutation strategies in the S100A6 promoter. WT, wild-type; Mut, mutant. (F) ChIP-qPCR products were visualized by agarose gel electrophoresis. (G) The efficiency of ChIP was calculated as percent input = 2% × 2(CT 2%Input Sample–CT IP Sample). (H) 500ng c-Myc overexpression plasmid or control plasmid was co-transfected with 500 ng plasmids containing S100A6 gene promoter/Firefly luciferase construct and 50 ng Renilla into 293T cells in the NG condition. Firefly and Renilla luciferase activities were measured 36 h after transfection. (I) 293T cells were cultured in the NG or HG condition for 5d and then co-transfected with plasmids containing S100A6 gene promoter/Firefly luciferase construct and 50 ng Renilla. Firefly and Renilla luciferase activities were measured 36 h after transfection. OE-NC, the negative control of c-Myc overexpression. shMyc-NC, the negative control of c-Myc knockdown. Data were shown as the mean ± SEM measured in triplicate from three independent experiments. ANOVA followed by Tukey’s multiple comparisons test were performed to analyze the differences. *P < 0.05, **P < 0.01.

Fig 3: S100A6 inhibits HaCaT differentiation (A–C) The expression of S100A6 was determined by immunofluorescence assay in HaCaT cells (A) or wound margin tissues of rats (B) and humans (C). The nuclei were stained blue using DAPI. Magnification: ×200, Scale bar =50 µm. The white dotted line represents the epidermis-dermis dividing line. (D) Morphology of undifferentiated and differentiated HaCaT cells was recorded. Magnification: ×100, Scale bar =25 µm. (E–G) The mRNA and protein expressions of c-Myc and S100A6 and protein expressions of TGM1, LOR and K1 were detected after the differentiation of HaCaT cells. (H) HaCaT cells was pre-treated with S100A6 recombinant protein (1 µM) for 24h and then TGM1, LOR and K1 protein levels were measured. (I, J) The mRNA and protein levels of TGM1, LOR, and K1 were determined after knocking down S100A6 in HaCaT cells. (K) TGM1, LOR, and K1 expression levels were determined in S100A6-knockdown HaCaT cells cultured with high glucose. (L) TGM1, LOR, and K1 expression levels were determined in HaCaT cells overexpressing c-Myc or meanwhile knocking down S100A6. shS100A6-NC, the negative control of S100A6 knockdown. NC, negative control. Data were shown as the mean ± SEM measured in triplicate from three independent experiments. ANOVA followed by Tukey’s multiple comparisons test were performed to analyze the differences. *P < 0.05, **P < 0.01.

Fig 4: Correlation matrix of the S100A6, MMP-9 and CST4 biomarkers (in bold) in the tear samples and the clinical variables of all the individuals. The r value for each correlation is shown in the squares, and the color scale indicates the degree of significance and its direction (negative in blue, positive in red).

Fig 5: Tear S100A6 (A), MMP-9 (B) and CST4 (C) concentrations (ng/mL) in the groups of patients suffering SS relative to the healthy controls (CTs). Cliff’s delta values are displayed for each comparison. In addition, statistical significances are displayed: *, p < 0.05; **, p < 0.01.