Fig 1: UNC2025, an inhibitor of MerTK, alleviated DSS-induced intestinal fibrosis(A) Western blot and grayscale analysis of MerTK and OPN in experimental mice with acute colitis or chronic fibrosis (n = 3).(B) qPCR evaluation of MerTK, OPN, Gas6, TGF-β1, COL1A1, and COL3A1 in mice (n = 3).(C) Masson’s trichrome staining of the colon in mice (n = 3).(D) Protein evaluation by western blotting of the MerTK pathway and OPN in different groups and corresponding grayscale analysis (Control: mice treated with tap water; DSS: 64-day 2% DSS-treated mice; DSS + UNC2023-D0: mice treated with 3 mg/kg UNC2025 from the first day; 2% DSS + UNC2025-D21: mice treated with 3 mg/kg UNC2025 from day 21; 2% DSS + UNC2025-D42: mice treated with 3 mg/kg UNC2025 from day 42; n = 3).
Fig 2: OPN regulated TGF-β1 secretion induced by MerTK activation(A and B) Evaluation of MerTK and OPN by western blotting, grayscale analysis (compared to the control group), and qPCR in intestinal specimens from patients with CD.(C) Transcriptional level of OPN in RAW 264.7 cells after 1 h of 400 ng/mL Gas6 stimulation.(D) Intracellular signal intensity of OPN in RAW 264.7 cells with or without 1-h Gas6 stimulation for immunofluorescence analysis.(E) Immunofluorescence staining and colocalization of MerTK and OPN in human specimens.(F) Changes in the MerTK pathway after knockdown of OPN with siRNA for western blotting and corresponding grayscale analysis (compared to the control group).(G) Transcriptional change (compared to the control group) in the MerTK pathway and TGF-β2, TGF-β3, and PDGF after knockdown of OPN or overexpression with plasmid transfection for qPCR analysis (control: vector transfected; siOPN: small interfering OPN RNA transfected).(H) Changes in the MerTK pathway after OPN overexpression with plasmid transfection for western blotting and grayscale analysis (compared to the control group).(I) TGF-β1 ELISA test for supernatants of RAW 264.7 cells (compared to the control group). All in vitro experiments were repeated at least three times to minimize technical and biological variability.
Fig 3: Profibrotic activity of MerTK+ macrophages in vitro(A and B) qPCR analysis for MerTK, OPN, TGF-β1, TGF-β2, TGF-β3, and PDGF in RAW264.7 cells after stimulation with 400 ng/mL Gas6.(C) Protein expression evaluation of MerTK and OPN in RAW264.7 cells by western blotting and grayscale analysis (compared to the control group) after stimulation with 400 ng/mL with 0, 30 min, 1 h, and 2 h.(D) Wound healing analysis in L929 cells cultured in the supernatant of RAW 264.7 cells with or without Gas6 stimulation.(E) Quantitative analysis of scratch area in the wound healing experiment by ImageJ.(F) Protein expression evaluation and grayscale analysis (compared to the control group) of MerTK/ERK/TGF pathway after knockdown of MerTK by transfection of siRNA into RAW 264.7 cells with or without Gas6 stimulation; (G) transcriptional evaluation of COL1A1 (left panel) and COL3A1 (right panel) of L929 cells after treatment with the supernatant of RAW264.7 cells. All in vitro experiments were repeated at least three times to minimize technical and biological variability.
Fig 4: Alectinib treatment resulted in an increase in the number of macrophages and resulting in elevated Gas6 levels in the tumor microenvironment (TME). (A) Bar plots showing the proportion of macrophages in samples collected from days 2 to 8 (left). Pie chart illustrating the proportion of immune cells in the day 8 samples. The mean proportions from triplicate samples are shown (right). B Cells Naïve, naïve B cells; DC Immature, immature dendritic cells; NK Activated, activated natural killer cells. (B) t‐SNE plots of 23,920 cells colored by sample origin (left) and 10 clusters determined using the k‐means method (right). Each cluster was annotated on the basis of differentially upregulated genes and expression levels of cell markers. “Veh_d8_1” represents replicate 1 of vehicle‐treated sample collected on day 8. Ale, alectinib; Veh, vehicle. (C) t‐SNE plots illustrating the expression patterns of Gas6 (left) and violin plots showing the Gas6 expression levels (log2 UMI count) in each cluster (right). (D) t‐SNE plots of cluster 4 colored by treatment group (left), violin plots showing the Gas6 expression (log2 UMI count) in each group (middle), and a pie chart illustrating the proportion of each group (right). *p < 0.05 (unpaired t‐test)
Fig 5: Gilteritinib was effective in overcoming resistance to ALK‐TKI treatment resulting from the activation of the GAS6/AXL signaling pathway. (A, B) Bar graphs showing the effects of gilteritinib (100 nmol/L) on the viability of H3122 parental (PT) and AXL‐overexpressing (AXL) cells. Recombinant GAS6 (400 ng/mL) (A) or conditioned medium from parental (PT) and single‐clone (#37) GAS6‐overexpressing (GAS6) NIH3T3 cells (B) was added 6 h before drug treatment. Cell viability was evaluated after 72 h using the CellTiter‐Glo Assay. (C) Immunoblot analysis of ALK, AXL, PARP, and the downstream pathways of ALK in parental (PT) and AXL‐overexpressing (AXL) H3122 cells. The cells were treated with the indicated concentrations of the drugs (Ale, alectinib; Gil, gilteritinib) for 6 h. Recombinant GAS6 (100 ng/mL) was added 1 h before the initiation of drug treatment.
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