Fig 2: PH domain in kindlin-3 is required for αLβ2 (LFA-1) clustering. A-F, Representative STORM images of integrin αL on differentiated HL60-2 cells containing wildtype kindlin-3 (WT-K3, A-C) and PH domain-deleted kindlin-3 (ΔPH-K3, D-F) without (A, D) or with IL-8 (B, E) or fMLP (C, F) stimulation after clustering analysis using Voronoi diagrams. Adjacent clusters are distinguished by different colors. Non-clustered αL are shown as gray dots. Scale bars are 1 μm. G-H, The number of clusters per WT-K3 or ΔPH-K3 HL60-2 cell without or with IL-8 (g) or fMLP (H) stimulation. Means ± SD (n ≥ 75 cells from 3 individual experiments). ns p > 0.05; ** p < 0.01; **** p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test

Fig 3: Kindlin-3 and talin-1 are essential for β2 integrin activation. Wildtype (WT), kindlin-3 knockout (K3-KO), talin-1 knockout (TLN1-KO), and β2 integrin knockout (β2-KO) CXCR2-expressing HL60 (HL60-2) cells were differentiated to neutrophil-like cells. A-D, β2 integrin activation was quantified by flow cytometry using conformation-specific antibodies mAb24 (A, C) and KIM127 (B, D) with or without IL-8 (A-B) or fMLP (C-D) stimulation. E-F, Total β2 integrin expression was quantified by flow cytometry using antibody TS1/18 with or without IL-8 (E) or fMLP (F) stimulation. G-H, Expression of CXCR2 and FPR1 on cells. MFI, median fluorescence intensity. Means ± SD, n = 12 repeats from 3 individual experiments in A-D and G-H, n = 16 repeats from 4 individual experiments in E-F. ns p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by two-way ANOVA in A-F and one-way ANOVA in G-H followed by Tukey’s multiple comparison test

Fig 4: Kindlin-3 but not talin-1 is important for αMβ2 integrin (Mac-1) clustering. A-I, Representative STORM images of integrin αM on wildtype (WT, A-C), K3-KO (D-F), and TLN1-KO (G-I) differentiated HL60-2 cells without (A, D, G) or with IL-8 (B, E, H) or fMLP (C, F, I) stimulation after clustering analysis using Voronoi diagrams. Adjacent clusters are distinguished by different colors. Non-clustered αM were shown as gray dots. Scale bars are 1 μm. J-M, The number of clusters per WT, K3-KO (J-K), or TLN1-KO (L-M) HL60-2 cell without or with IL-8 (J, L) or fMLP (K, M) stimulation. Means ± SD (n ≥ 50 cells from 3 individual experiments). ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test

Fig 5: Kindlin-3 but not talin-1 is critical for αLβ2 integrin (LFA-1) clustering. A-I, Representative STORM images of integrin αL on wildtype (WT, A-C), kindlin-3 knockout (K3-KO, D-F), and talin-1 knockout (TLN1-KO, G-I) differentiated HL60-2 cells without (A, D, G) or with IL-8 (B, E, H) or fMLP (C, F, I) stimulation after clustering analysis using Voronoi diagrams. Adjacent clusters are distinguished by different colors. Non-clustered αL are shown as gray dots. Scale bars are 1 μm. J-M, The number of clusters per WT, K3-KO (J-K), or TLN1-KO (L-M) HL60-2 cell without or with IL-8 (J, L) or fMLP (K, M) stimulation. Means ± SD (n ≥ 50 cells from 4 individual experiments). ns, p > 0.05; * p < 0.05; ** p < 0.01; **** p < 0.0001, by two-way ANOVA followed by Tukey’s multiple comparison test