Fig 1: Identification of CEGR/ALCD-containing genes that are elevated in patients with FLC. (A) RNA-sequencing (RNA-seq) results for patients with FLC who have elevated DNAJB1-PKAc-ß-catenin-CEGRs/ALCDs pathways. Red colors show mRNAs coding for components of the DNAJB1-PKAc-ß-catenin-TCF4 axis. (B) Sequences of CEGRs/ALCDs in the COL4A1, SPARC, and VCAN genes. (C) Confirmation of RNA-seq results by quantitative real-time PCR. (D) Expression of up-regulated proteins detected by western blotting. (E) ChIP assay performed with CEGRs/ALCDs for HDAC1, DNAJB1, and SPARC genes.
Fig 2: DNAJB1-PKAc-ß-catenin-TCF4-CEGRs/ALCDs pathway is preserved in lung metastasis of a patient with FLC. (A) Expression of the fusion DNAJB1-PKAc transcript in liver tumor and lung metastasis. (B) Hematoxylin and eosin (H&E) staining, sirius red staining, and immunostaining of primary hepatic tumor (upper) and lung metastasis (bottom) of the patient with FLC with antibodies to PKAc and ph-S675-ß-catenin. The ×20 insert shows nuclear staining of cells with ph-S675-ß-catenin. Cells with nuclear (red arrows) and cytoplasmic (black arrows) staining are shown. (C) Examples of ph-S675-ß-catenin-positive cells with mitotic figures are observed in lung metastasis of a patient with FLC. (D) Western blotting shows the expression of proteins of the ß-catenin-TCF4-p300 complexes in lung metastasis. Right: Co-IP studies. (E) Western blotting shows the expression of CEGR/ALCD-dependent genes in lung metastasis. (F) ChIP assay of the CEGRs/ALCDs from DNAJB1, HDAC1, and SPARC genes. (G) Hypothesis that is based on the studies of primary tumors and a lung metastasis of patients with FLC.
Fig 3: Sparc transcription decreases during inflammatory conditions. (A) Total RNA was obtained from myoepithelial cells isolated from lacrimal glands of SMA–GFP mice. The cells were cultured with IL-1β, TNF-α, or vehicle control. The relative levels of Sparc expression were determined using qPCR (n = 3–6 independent experiments). (B) H&E staining of the exorbital lacrimal glands of control 16-week-old BALB/c mice and 16-week-old NOD mice. Lacrimal glands from BALB/c mice show normal morphology with lobules devoid of immune cells. In contrast, lacrimal glands from NOD mice are heavily infiltrated by immune cells that formed large foci within the lobules (arrows). Scale bar: 100 µm. Total RNA isolated from the lacrimal glands of these mice was used to determine the relative levels of Sparc expression using qPCR (n = 6 mice/group). The box-and-whisker plots show the 25th and 75th percentiles (boxes), the median, and the minimum and maximum data values (whiskers). Significance was determined using the non-parametric Kruskal–Wallis test with Dunn's test for multiple comparisons (A) or the Mann–Whitney test (B). *P < 0.05; **P < 0.01.
Fig 4: SPARC is downregulated in the adult lacrimal gland. (A) Total RNA was isolated from exorbital lacrimal glands from C57BL/6J mice at different ages (4–6 weeks old, 17–18 weeks old, and 32–40 weeks old; n = 5 or 6 mice per group). The relative levels of Sparc expression were determined using qPCR. (B) Total protein extracts were prepared from homogenized exorbital lacrimal glands from young (4–6 weeks old) and adult (32–40 weeks old) C57BL/6J mice and analyzed using ELISA (n = 8 mice/group). The box-and-whisker plots show the 25th and 75th percentiles (boxes), the median, and the minimum and maximum data values (whiskers). Significance was determined using the non-parametric Kruskal–Wallis test with Dunn's test for multiple comparisons (A) or the Mann–Whitney test (B). *P < 0.05; **P < 0.01.
Fig 5: Methylation state of CpG sites in the Sparc gene promoter region. The gDNA was isolated from exorbital lacrimal glands (n = 6 mice/group) from young (4–6 weeks old) and adult (32–40 weeks old) C57BL/6J mice. The methylation of four specific CpG sites in the Sparc gene promoter region (-208, -171, -135, and -101) was evaluated by bisulfite genomic sequencing after PCR amplification and cloning into a vector. The graph shows the percentage of methylated and unmethylated sites in each group of mice, as well as the total number of sequences analyzed (in parentheses).
Supplier Page from Abcam for Anti-SPARC antibody