Fig 2: Interaction of HSP70 and KANK2 in A549 cells. WT and HSP70OE A549 cells were treated with LPS (4 µg/mL) and si-KANK2 (50 nM, si-NC was set as corresponding control). Cell viability (A) was measured by CCK-8 and cell colony formation (B,G) was evaluated by crystal violet staining. Dead cells were enumerated by flow cytometry labelling with Annexin V and PI (C,H). The levels of KANK2 and released AIF were measured using WB (D,E). Mitochondrial membrane permeability was assessed by mito-tracker and fluorescence intensity was measured by imaging system (F). Data are presented as mean ± SD of data from three independent experiments. Bar = 200 µm. ** p < 0.01.

Fig 3: RNA sequencing and proteomics in HEK 293T cells and validation in A549 cells. HEK 293T cells were transfected with hsp70-plasmid. Transcriptomes analysis was conducted in HEK 293T cells by RNA sequencing. Heat map of the cluster analysis (A) and volcano plot (B) was presented. The protein binding to HSP70 was extracted by IP (anti-Flag-HSP70). Then, the protein was separated by electrophoresis and stained with silver (C). Proteomics analysis was conducted by MS. Enrichment analysis for the binding protein was shown (D). IP was performed with an anti-Flag antibody. The expressions of HA-KANK2 and Flag-HSP70 were measured in HEK 293Ts. KANK2 bands were indicated with black arrow (E). A549 cells were cultured. The mRNA (F) and protein (G) levels of KANK2 in HSPOE A549 cells were measured using qPCR and WB. IP was performed with an anti-HSP70 antibody, and KANK2 was assessed in A549 cells (H). Representative immunofluorescence images of HSP70 and KANK2 in the A549 cells were acquired (I). Bar = 10 µm. BP, biological process; CC, cellular component; MF, molecular function; MS, mass spectrometry. ** p < 0.01.