Fig 1: CXCL8 functions in the eHsp90a-induced lymphangiogenic process in hLECs. (A) The transcriptome levels of hLECs in the Hsp90a Ab group and the IgG control group were analyzed. Volcano plot was drawn to present the upregulated and downregulated DEGs by Hsp90a neutralizing antibody treatment. In total, 59 upregulated DEGs and 118 downregulated DEGs were colored in red and blue, respectively. (B) Venn diagram was drawn to show the overlapping genes between upregulated DEGs and negative-related lymphangiogenic genes, also between downregulated DEGs and positive-related lymphangiogenic genes. (C) Validation of RNA-seq results by qRT-PCR. (D) Further exploration of CCL24, CXCL8, HDAC9 expression in hLECs treated with recombinant Hsp90a protein by qRT-PCR. (E) Immunoblotting analyses of CXCL8 expression stimulated by recombinant Hsp90a protein in the presence of Hsp90a neutralizing antibody or control IgG. Quantitative results are shown below. (F) The protein level of CXCL8 induced by recombinant Hsp90a protein with or without MK-2206. Quantitative results are shown below. (G) Quantification of secreted CXCL8 levels in hLEC supernatant measured by ELISA. (H,I) Representative and quantified data showing the relative migration (H) and tube formation (I) activities of hLECs stimulated with recombinant Hsp90a protein (200 ng/mL) or CXCL8 protein (1 ng/mL) in the presence or absence of CXCL8 neutralizing antibody (10 ng/mL). Scale bar = 200 µm. Data are shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Fig 2: The expression of mRNA and protein of PML-RARa, AML1-ETO, CXCL8, CDKN1A, and CCND2 related to the cell differentiation or cell cycle arrest. (A,B) Real-time PCR and Western blotting were used to determine the mRNA and protein expression in NB4 and Kasumi-1 cell lines incubated with OGP46. NB4 or Kasumi-1 cells were treated with OGP46 (1 µM) with or without ATRA (1 µM) or Ara-C (1 µM) for 96 h for detection of PML-RARa and AML1-ETO. NB4 or Kasumi-1 cells were treated with OGP46 (1 µM), ATRA (1 µM), or Ara-C (1 µM) for the indicated time for detection of CXCL8, CDKN1A, and CCND2. (C) Protein expression was quantified by the software AI600 images. *p < 0.05 and **p < 0.01.
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