Fig. 6: Hemocytin (hmc) positively impacts the JNK pathway in I. scapularis.

a, b Tick cells were transfected with either hmc siRNA (si-hmc) or scrambled RNA (sc-hmc). a Efficiency of hmc silencing in IDE12 cells (n = 12 and 11). b Representative western blot (left) of N-Rel and p-JNK during treatment with sc-hmc (lane 1) or si-hmc (lane 2). N-Rel and p-JNK protein expression was quantified (right) in si-hmc (blue) or sc-hmc (grey) IDE12 cells (n = 4). Data were normalized to the scrambled control, with N-Rel values relative to Actin and p-JNK values to JNK. A representative blot from four experiments is shown. Uncropped blots containing the molecular weight markers are supplied in the Source data. c Overview of CRISPRa-mediated hmc overexpression in ISE6 cells. d RT-qPCR analysis of hmc (left; n = 9 and 10), jnk (middle; n = 9 and 9) and jun (right; n = 10 and 10) expression in dCas9⁺ ISE6 cells transfected with either a single guide RNA (sgRNA) specific to the promoter region of hemocytin (hmc-sgRNA, blue) or a random sgRNA (ctrl-sgRNA, grey). e–h Ticks were subjected to microinjection with hmc siRNA (si-hmc; blue) or scrambled siRNA (sc-hmc; grey) and subsequently fed on either (e–g) uninfected or (h) A. phagocytophilum-infected mice. e RT-qPCR analysis of hmc (left; n = 19 and 18), relish (middle; n = 17 and 18) and jun (right; n = 17 and 18) expression in engorged ticks. f Weight measurements of engorged nymphs (n = 20 and 23). g Percentage of nymphs that molted to adults. h RT-qPCR assessment of hmc silencing efficiency (n = 11 and 10) and A. phagocytophilum load (n = 11 and 10) in individual infected ticks. Bacterial quantification was based on the expression of A. phagocytophilum 16 s rRNA (Ap16S) gene. Results are represented as mean ± SD. A minimum of two independent experiments were performed. Statistical significance was evaluated by (a, b, d, e) an unpaired two-tailed t-test with Welch’s correction; (f, h) a two-tailed Mann–Whitney U test or (g) by a Fisher exact test, and significant p-values (< 0.05) are displayed in the figure. Source data are provided as a Source Data file. N-Rel = cleaved Relish; p-JNK = phosphorylated JNK; JNK = c-Jun N-terminal kinase.