Fig 1: LAMP3 expression in salivary gland epithelial cells is involved in BMP6 expression by the release of HSP70.(A) Scatter plot showing enriched gene ontology in patients with SS with high BMP6 expression compared with those with normal BMP6 expression. (B) Correlation between LAMP3 and BMP6 transcript expression in minor salivary glands of patients with SS (n = 43). (C) Schematic showing the methods used in the in vitro assays. (D) Representative Western blot with the indicated antibodies using lysates of salivary acinar or ductal cells 72 hours after transfection with empty and/or LAMP3 expression plasmids. (E) HSP70 concentration in culture supernatant collected 96 hours after transfection. (F) THP1 cells were treated with the culture supernatant of acinar or ductal cells with or without CUCPT22 (20 µM) or TAK242 (40 µM). BMP6 transcript levels in THP1 cells were evaluated 20 hours after stimulation using the ??Ct method relative to ACTB. Values shown are the mean ± SEM of 3 independent experiments. **P < 0.01, by Student’s t test with multiple testing correction using Tukey’s method (E and F).
Fig 2: HSP70 is released by caspase-dependent lysosomal exocytosis from LAMP3-overexpressing epithelial cells.(A–H) HSG cells were transfected with empty and/or LAMP3 expression plasmids and then treated with vacuolin-1 (at the indicated concentration), ZVAD (20 µM), ZDEVD (10 µM), or YVAD (50 µM). Lysosomal exocytosis was monitored by quantifying LAMP1 expression on the cell surface, and the intracellular Ca2+ concentration was determined by Fluo-4 fluorescence by flow cytometry 48 hours after transfection. (I) The HSP70 concentration was evaluated in culture supernatant collected from HSG cells 72 hours after transfection with or without ZVAD (20 µM) or vacuolin-1 (10 µM). Values shown are the mean ± SEM from 3 (I) or 4 (A–H) independent experiments. **P < 0.01, by Student’s t test with multiple testing correction using Tukey’s method (B) or Dunnett’s method (D, F, H, and I). Max, maximum.
Fig 3: BMP6 expression is upregulated via the TLR4 pathway in human monocytes.Human PBMCs were treated with sham control, LPS (100 ng/mL), or HSP70 (1 µg/mL) for 20 hours and then analyzed using the 10× Genomics platform. (A) Clustered PBMCs are displayed in UMAP format with the distribution of cells expressing BMP6. (B) Relative expression of BMP6 and the number of BMP6+ cells in each group. (C) Proportion and relative expression of the indicated genes in each cell cluster. (D) Connectivity and trajectory among cell clusters. (E) Top 20 DEGs between BMP6+ monocytes and CD14++CD16– (classical) monocytes. (F) General differentiation pathway of the monocyte lineage.
Fig 4: HSP70 stimulates BMP6 expression via the TLR4/MyD88 pathway.(A) Venn diagram showing increased expression of potential ligands for TLR4 (listed inside the largest oval) in minor salivary glands (yellow oval), saliva (blue oval), and/or serum/plasma (shown in red) from patients with SS. (B) Serum HSP70 and BMP6 levels in patients with SS (n = 50) and HVs (n = 30). Boxes represent first, second, and third quartiles. ††P < 0.01, by Wilcoxon test. RFU, relative fluorescence units. (C and D) THP1 cells were treated with the indicated recombinant proteins at the indicated concentration for 4 hours. C, control. (E) THP1 cells were treated with boiled HSP70 (1 µg/mL) or LPS (100 ng/mL) for 4 hours. (F) THP1 cells were pretreated with TLR1/2 antagonist (CUCPT22, 20 µM) or TAK242 (40 µM) 1 hour prior to HSP70 simulation. (G) WT, MYD88–/–, or IRF3–/– THP1 cells were stimulated with HSP70 for 4 hours. TNFA and BMP6 transcript levels in the cells were quantified after each treatment using the ??Ct method relative to ACTB expression. Values shown are mean ± SEM of 3 independent experiments. **P < 0.01, by Student’s t test with multiple testing correction using Dunnett’s method (C–G).
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