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. 2021 Mar 4;11(10):4655–4671. doi: 10.7150/thno.49007

Figure 7.

Figure 7

IL-6-activated STAT3 was required for Tpo transcription. (A) The concentration of IL-6 in the plasma was examined by ELISA. The data shows one of two independent experiments (n = 5 - 6 mice/group) with similar results. **P < 0.01. (B) CD19+ B cells (3 × 104) in the spleens of the Aurkaf/f mice or the Aurkaf/f;Cd19Cre/+ mice were analyzed. The dot graph shows the total number of IL-6-positive cells in these CD19+ B cells. The data shows one of two independent experiments (n = 5 mice/group) with similar results. *, P < 0.05. (C, left) Immunofluorescence for CD19 (red), IL-6 (green) and DAPI (blue) in the BM sections from the Aurkaf/f or Aurkaf/f;Cd19Cre/+ mice. Mk, megakaryocyte. (C, right) Immunofluorescence for CD19 (red), TPO (green) and DAPI (blue) in the BM sections from the Aurkaf/f or Aurkaf/f;Cd19Cre/+ mice. The data shows one of two independent experiments with similar results. (D) Dot graph of Figure 7C shows the number of CD19+IL-6+ B cells, or CD19+TPO+ B cells close to megakaryocytes in the BM sections from the Aurkaf/f mice (n = 3 mice/group, 5 slides/mouse) or the Aurkaf/f;Cd19Cre/+ mice (n = 3 mice/group, 5 slides/mouse). *, P < 0.05. (E) Aurkaf/f mice were treated with either control diluent or 1 µg IL-6. After which, hepatocytes were isolated and subjected to real-time RT-PCR to quantify the Tpo mRNA levels. The data shows one of two independent experiments (n = 5 mice/group) with similar results. *, P < 0.05. (A-E) P-values were obtained using a 2-sided, unpaired Student's t-test. (F) The expression of the indicated protein was examined in spleens from either the Aurkaf/f or Aurkaf/f;Cd19Cre/+ mice by Western blotting. (G) CD19+ B cells were sorted from either the Aurkaf/f or Aurkaf/f;Cd19Cre/+ mice. 5 × 105/mL sorted CD19+ B cells were exposed to 10 mM C188-9. After 24 h, cells were harvested, fixed and subjected to FACS to examine the levels of p-STAT3. (F-G) The data shows one of two independent experiments. (H) CD19+ B cells were sorted from either the Aurkaf/f or Aurkaf/f;Cd19Cre/+ mice. 1 × 106/mL sorted CD19+ B cells were exposed to 10 mM C188-9. After 24 h, cells were harvested, RNA was extracted and subjected to real-time RT-PCR to quantify the Tpo mRNA levels. Data shown is mean ± SD of one of two independent experiments (n = 3 mice/group) with similar results. Significance was calculated using an unpaired Student's t-test. *, P < 0.05. (I) The correlation of STAT3 and TPO was assessed by GEPIA. (J) CD19+ B cells were sorted from either the Aurkaf/f or Aurkaf/f;Cd19Cre/+ mice. After being crosslinked with formaldehyde, protein-DNA complexes were immunoprecipitated using an anti-STAT3 antibody. After which, DNA was extracted and subjected to PCR to measure the STAT3 binding sites on the TPO promoter. The PCR products were separated in a 2% agarose gel and visualized by ethidium bromide staining. bp, base pair. Data shows one of two independent experiments with similar results. (K) CD19+ B cells were sorted from either the Aurkaf/f or Aurkaf/f;Cd19Cre/+ mice. 1 × 106/mL sorted CD19+ B cells were exposed to either 10 mM C188-9 and/or 100 ng/mL IL-6. After 24 h, cells were harvested, RNA was extracted and subjected to real-time RT-PCR to quantify the Tpo mRNA levels. Data shown is mean ± SD of one of two independent experiments (n = 3 mice/group) with similar results. Significance was calculated using an unpaired Student's t-test. *P < 0.05.