Fig 2: ILK Induces Hsc70 and Hsp70 Expression in Human Cardiomyocytes. A, (top panels) Phase contrast and fluorescent microscopy images of human IPSC-derived cardiomyocytes (Hu iPS-CMs) (on the left) and primary rabbit cardiomyocytes (on the right) transduced with GFP-linked vector (ad-GFP) versus non transduced cells (C). The Hu iPS-CM cells express monomeric red fluorescent protein and blasticidin resistance under the control of the α-myosin heavy chain promoter that allows simultaneous cardiomyocyte purification and identification. Green fluorescence indicates efficient transduction of ILK constructs. Scale bar, 80 µm. (bottom panels) Western blots showing expression levels of ILK, Hsp70 and Hsc70 in Hu iPS-CMs (on the left) and in primary rabbit cardiomyocytes (on the right) transduced with ILKWT and ILKR211A as compared to non-transduced cells and cells transduced with the vector bearing GFP only. GAPDH was used as a loading control. B, Immunoblot for ILK expression levels in Hu IPS-CMs transduced with ad-ILKWT and ad-ILKR211A as compared to non-transduced cells following treatment with cycloheximide (CHX) at 80 µM for 0, 3, 6 and 18 hours. C, Western blot analysis showing the protein levels of ILK, Hsp70 and Hsp-90 in Hu iPS-CMs transduced with ad-GFP, ad-ILKWT, ad-ILKR211A and in non-transduced cells (C) following treatment with indicated doses of Hsc/p70-ATPase inhibitor (Ver-155008) for 24 hours. GAPDH was used as a loading control. Each experiment was performed at least three times on independent samples and one representative blot is shown.

Fig 3: Overexpression of the E359K mutant does not reconstitute CD cell function.A, CD cells with similar levels of WT and E359K-ILK (E359K/Lo) or 2.5 times the amount or E359K (E359K/Hi) were generated. The solid white line represents lane splicing from the same gel. B, the relative expression of ILK in the cells is depicted graphically. (C) cell adhesion, (D) migration, and (E) proliferation were performed as described in Figure 2. ∗∗p < 0.01 between WT and E359K and #p < 0.05 between E359K Hi and E359K Lo.

Fig 4: Phospholipid peroxidation-caused 4-HNE modification of ILK dampens RUNX2 signaling.a Workflow for immunoprecipitation-MS-based proteomic. b Volcano plots of LC-MS identified proteins in RSL3-treated group and control group. (log2 (fold change)) versus significance (-log10 (P value)) by unpaired two-tailed t-test. The significance in the volcano plot is defined by a fold change of more than 4 times and a P-value less than 0.01. c, d Co-IP assay determined the level of ILK conjugated with 4-HNE in MC3T3-E1 cells treated with RSL3 (50 nM, 24 hours). e Representative confocal images showing the colocalization of 4-HNE (green) and ILK (red) in MC3T3-E1 cells treated with RSL3 (50 nM, 6 hours). Scale bars = 10 μm. f ILK expression in MC3T3-E1 cells treated with RSL3 for 24 hours (n = 3 independent cell culture). g Ilk mRNA expression in MC3T3-E1 cells treated with RSL3 for 24 hours (n = 3 independent cell culture). h Turnover of ILK by CHX chase assay in MC3T3-E1 cells treated with or without 4-HNE (n = 3 independent cell culture). CHX, 100 μM; 4-HNE, 10 μM. i The effect of MG132 (5 μM) or 3-MA (20 μM) on the RSL3 (50 nM)-induced ILK protein level (n = 3 independent cell culture). j Ubiquitination of ILK assessed by immunoblotting in MC3T3-E1 cells with RSL3 treatment (50 nM, 24 hours). The experiment was repeated three times independently with similar results. k ILK and RUNX2 expression in MC3T3-E1 cells after ILK knockdown (n = 3 independent cell culture). l Bone-forming genes expression in MC3T3-E1 cells after ILK knockdown (n = 3 independent cell culture preparations). m ALP staining in 1-week osteogenic-induced MC3T3-E1 cells after ILK knockdown. n The verification of ILK overexpression. o Bone-forming genes expression in MC3T3-E1 cells treated with RSL3 and ILK overexpression plasmid (RSL3, 50 nM, 24 hours) (n = 3 independent cell culture preparations). p Schematic diagram illustrating that phospholipid peroxidation-derived 4-HNE binds to ILK, resulting in ILK degradation, disturbing bone formation. Data are presented as means ± SD. Statistical analysis is performed with one-way ANOVA with Tukey’s post hoc test (f, g, i, k, o) or two-way ANOVA (h). Source data are provided as a Source Data file.

Fig 5: ILK expression in different cell types in colon cancer. ILK expression was examined in patient colon cancers via the immune cell atlas platform [Single Cell Portal (broadinstitute.org)]. (A) t-distributed stochastic neighbor embedding (t-SNE) plot showing B cells (B), epithelial cancer and non-cancer cells (Epi), myeloid cells (Myeloid), plasma cells (Plasma), and T and NK and innate lymphoid cells (TNKILC) of colon cancer. (B) ILK expression in all cell clusters. (C) FOXP3 expression for Tregs in all cell clusters. (D) CD163 expression for M2 macrophages in all cell clusters.