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. 2022 Oct 3;12(16):6989–7008. doi: 10.7150/thno.75069

Figure 4.

Figure 4

CHI3L1 binds to ACTN4 and NFKB1, and promote the activation of NF-κB pathway. (A) Schematic of the procedure used to detect biotin-hCHI3L1-binding proteins using HuProt 20K human proteome microarrays. (B) ACTN1, 4, NFKB1, 2, and NFKBIB were identified as CHI3L1-binding proteins in the proteome microarrays. (C) The IF analysis of the expression levels of CHI3L1 and NF-κB p65 subunit in peritumor and intratumor regions; Scale bars represent 100 µm. (D) Western blot analysis of the expression levels of CHI3L1 in U118MG and U251MG cells after treated with TNFɑ (200 ng/mL) for 0-96 h. (E) Chi3l1+/+ and Chi3l1-/- BMDMs were treated with TNFɑ (50 and 200 ng/mL), and phosphorylation of p65 were assessed via western blot. (F-G) U87MG and A172 cells transfected with shCtrl and shCHI3L1 were treated with TNFɑ, and the phosphorylation of p65 were detected using western blot. (H-I) Cytoplasmic and nuclear proteins were isolated from BMDMs and U87MG after treated with TNFɑ (50 ng/mL) and applied to western blot to detect the expression of p65 and ACTN4. (J) Lysates from U87MG and A172 cells were immunoprecipitated with IgG or anti-CHI3L1 antibody, and then immunoblotted as indicated. (K) Cytoplasmic and nuclear proteins were isolated from U87MG cells. The lysates were immunoprecipitated with IgG or anti-CHI3L1 antibody, and then immunoblotted as indicated. (L-N) Co-localization of CHI3L1 and ACTN4, NFKB1, or p65 in U87MG or A172 cells observed by confocal microscope. (O) CHI3L1 and ACTN4 enhance NF-kB activation by using a dual-luciferase reporter assay. (P) Dox-inducible CHI3L1 expression enhanced enhanced NF-kB activation by TNFα in U251 cells. (Q) Pearson correlations between CHI3L1 expression and ACTN4, NFKB1, NFKB2, RelA (p65), and NFKBIB in TCGA and CGGA glioma cohorts.