Fig 2: ITLN1 abrogates LTF’s effects on ovarian cancer cells’ motility.a Western blot analysis shows ITLN1 on proteins pulled down by an anti-LTF antibody. Normal mouse IgG served as a control. ITLN1 band size = 34 kDa. b Representative microscopic images from a Duolink proximity ligation assay (PLA) on SKOV3 and A224 shows the interaction between LTF and LRP1 while ITLN1 reduced the interaction. Red fluorescent signals indicate protein–protein interaction; nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) blue. Staining with no primary antibody and with anti-LTF antibody plus normal rabbit IgG served as controls. Bar = 5 µm. Representative microscopic images show that LTF induced c cell migration ability and d cell invasion potential in SKOV3 and A224 compared with control cells, while ITLN1 counteracted the effect. Bar = 50 µm. e Bar charts show that LTF upregulated the relative MMP1 mRNA expression in SKOV3 and A224, while ITLN1 counteracted the effect. f Western blot analysis shows that LTF upregulated MMP1 protein expression in SKOV3 and A224, while ITLN1 counteracted the effect. ß-actin served as a loading control. Relative normalized protein levels with respect to the corresponding control are presented. Three independent experiments were performed. b, e Results from three independent experiments were averaged and are shown as mean ± SD (two-tailed t-test). n. s. not significant (p > 0.05).

Fig 3: ITLN1 inhibits the LTF/LRP1/MMP1 signaling pathway.Western blot analyses show (a) higher protein levels of p-ERK1/2, p-Jun (S73), total Jun, and MMP1 in LTF-treated (100 µg mL-1) SKOV3 and A224 compared with control cells without treatment; b lower protein levels of p-ERK1/2, p-Jun (S73), total Jun, and MMP1 in LTF-treated SKOV3 and A224 with ITLN1 (500 ng mL-1) compared with control cells without ITLN1; and c lower protein levels of LRP1, p-ERK1/2, p-Jun (S73), total Jun, and MMP1 in LTF-treated SKOV3 and A224 with LRP1-specific siRNAs compared with control cells with negative siRNA. ß-actin served as a loading control. Relative normalized protein levels with respect to the corresponding control are presented. Three independent experiments were performed.

Fig 4: ITLN1 suppresses ovarian cancer progression via metabolic shift in vivo.a Images show a decrease in bioluminescence of IG10 cell-bearing C57BL/6 mice with ITLN1 treatment (n = 10) compared with untreated controls (n = 10) 6 weeks after treatment. Box plot shows a significantly (b) smaller tumor growth rate in; c smaller volume of ascites collected from; d higher ITLN1 concentration in serum collected from; and e lower glucose concentration in whole blood collected from IG10 cell-bearing C57BL/6 mice with ITLN1 treatment (n = 10) compared with untreated controls (n = 10) (p = 0.007, p = 0.015, p = 0.005, and p = 0.004, respectively; Mann–Whitney U test) 6 weeks after treatment. f Representative microscopic images of paraffinized sections of tumor tissues collected from IG10 cell-bearing C57BL/6 mice 6 weeks after treatment shows a significantly lower MMP1 expression in the ITLN1 treatment group compared with the untreated controls. Bar = 10 µm. Quantification of the staining intensity for each group (n = 10) is shown in dot plot. p = 0.001; Mann–Whitney U test. g Representative images show reduced intensity of hexose-6-phosphate (glucose-6-phosphate and fructose-6-phosphate) (ion adduct: [M-H]; mass to charge ratio (m/z): 259.0224) in tumor cells (white circles) in frozen omental tumor tissue sections from 0 to 1 h post injection of ITLN1, while increased intensity of hexose-6-phosphate is seen in adjacent adipocytes (black circles) post injection of ITLN1. The same tissue sections were stained with haematoxylin and eosin (H&E) after MALDI-imaging mass spectrometry (IMS). T tumor cells, A adipocytes; Bar = 200 µm. Three ROIs for each region (tumor and adipocyte) were randomly selected for each section (n = 5) based on histology of cell types for the analysis. h Box plot shows the normalized hexose-6-phosphate intensity in tumor cells (upper panel) and adipocytes (lower panel) at different time points after ITLN1 was intraperitoneally injected into C57BL/6 mice. (p = 0.002 and 0.001, respectively; Kruskal–Wallis H test). n = 5 in each group. a–e, h In the box plot, the boxes represent the interquartile range of the records, and the lines across the boxes indicate the median value of the records. The whiskers indicate the highest and lowest values among the records that are no more than 1.5 times greater than the interquartile range.

Fig 5: UVB-induced expression of photoaging-related genes was restored by the administration of MAPK inhibitors as well as kaempferide (KFD). NIH-3T3 cells were irradiated with UVB in the presence or absence of either ROS inhibitor: NAC, MAPK inhibitors (SB203580: p38 inhibitor, SP600125: JNK inhibitor, and PD98059: MEK inhibitor), PI3K inhibitor: LY294002, or KFD (5 µM). The value of (A) MMP-1a, COL1A1, (B) MCP-3, IL-8 and IL-6 was estimated by qRT-PCR. Representative results of at least three independent experiments are shown. All data are expressed as the mean ± standard deviation (SD) of three wells. Different letters (a–f) indicate the significant differences between groups (p < 0.05).