Fig 2: The molecular mechanism involved in lncRNA DLX6-AS1 affecting LCSCs by regulating STAT3 signaling pathway through affecting CADM1 promoter methylation. Down-regulation of lncRNA DLX6-AS1 inhibited CADM1 promoter methylation, increased CADM1 expression, and suppressed the activation of the STAT3 signaling pathway, and finally the expression of CD133 and CD13 in LCSCs were decreased and the progression of LCSCs was repressed. lncRNA DLX6-AS1, long non-coding RNA DLX6-AS1; CADM1, cell adhesion molecule; STAT3, signal transducer and activator of transcription 3; LCSC, liver cancer stem cells

Fig 3: Reduced lncRNA DLX6-AS1 inhibits spheroid formation ability, colony formation ability, and proliferation ability of LCSCs via the inhibition of CADM1–mediated STAT3 signaling pathway. a, mRNA levels of LCSCs markers determined by RT-qPCR; b and c, protein levels of LCSCs markers determined by western blot analysis; d, spheroid formation ability of LCSCs detected by spheroid formation assay, scale bar = 100 µm; e, colony formation ability of LCSCs detected by colony formation assay, scar bar = 50 µm; f, proliferation ability of LCSCs determined by EdU staining, scale bar = 50 µm; *, p < 0.05, vs. the blank group; the statistical data were expressed as mean value of standard error and analyzed by one-way ANOVA; the experiment was conducted 3 times; CADM1, cell adhesion molecule 1; STAT3, signal transducer and activator of transcription 3; RT-qPCR, reverse transcription quantitative polymerase chain reaction; ANOVA, analysis of variance; LCSCs, liver cancer stem cells; lncRNA, long non-coding RNA; DLX6-AS1, DLX6 antisense RNA 1; CD133, prominin-1; CD13, aminopeptidase N

Fig 4: A. phagocytophilum engagement of CD13 elicits Src kinase and Syk phosphorylation. HL-60 cells were treated with isotype control or CD13781–967 antibodies and then incubated with A. phagocytophilum DC organisms. Uninfected HL-60 cells (U) were included as a control. Western blot and densitometric analyses were performed to detect phosphorylated and total levels of Src and Syk at the specified time points. (A) Western blots were probed for phospho-Src (Y416), Src, phospho-Syk (Y525/526), Syk, A. phagocytophilum P44, and GAPDH. Star indicates bands representative of phosphorylated Src. (B–D) The densitometric signals of phosphorylated Src, Syk, or P44 proteins normalized to that of GAPDH are presented. Data are representative of three independent experiments and presented as the mean ± SD. One-way ANOVA with Tukey’s post hoc test was used to test for significant differences among groups. Statistically significant values relative to isotype-treated cells are indicated (**, P < 0.01; ****, P < 0.0001).

Fig 5: CD13 is required for A. phagocytophilum to productively infect mice. CD13 knockout (KO; seven females and five males) or wild-type (WT; five females and six males) mice were intraperitoneally injected with A. phagocytophilum DC organisms. Peripheral blood samples drawn on the indicated days were examined by light microscopy for A. phagocytophilum-infected neutrophils. Each symbol corresponds to the percentage of infected neutrophils as determined by examining at least 100 neutrophils per mouse. Two-way ANOVA with Tukey’s post hoc test was used to test for significant differences among groups. Data are presented as the mean ± SD. Statistically significant values are indicated (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).