SHKBP1 decreases the fluidity of cellular p62 bodies. (A) WT and SHKBP1 KO HeLa cells were transfected with GFP-p62 alone or in combination with mScarlet-i-SHKBP1. 24 h later, cells were subjected to live-cell imaging by confocal microscopy and analysis by FRAP. Shown is a representative image for each condition. A prebleach image is provided along with a time course of postbleach images (postbleach time indicated in min). Scale bars: 2 μm. (B) Quantification of the fluorescence recovery rates of GFP-p62 from FRAP experiments. Data were analyzed with GraphPad Prism using nonlinear regression (curve fit), shown as mean ± standard deviation (SD). n = 3 for each group from three independent experiments. (C) Quantification of the half-life of fluorescence recovery of GFP-p62 from FRAP experiments. n = 20 for each group from three independent experiments. Exact P values indicated from one-way ANOVA with Tukey’s post hoc test. (D) Western blot analysis of the whole-cell lysates (WCL) from three HEK293T cell lines stably expressing endogenously tagged p62 using mNG: SHKBP1 WT, SHKBP1 KO, and SHKBP1 WT with transient overexpression (OE) of a large amount of FLAG-SHKBP1. The hollow arrowhead indicates the FLAG-SHKBP1, and the solid arrowhead indicates untagged (endogenous) SHKBP1. (E) Western blot analysis of WCL, FT, and anti-mNeonGreen IP to assess the interaction between SHKBP1 and endogenously tagged p62 in a HEK293T cell line stably expressing mNG-p62 and transfected with a small amount of FLAG-SHKBP1. For the p62 blot, asterisks denote background bands from p62 antibody and the solid arrowhead indicates mNG11-tagged p62. For the SHKBP1 blot, hollow arrowhead indicates the FLAG-SHKBP1, and the solid arrowhead indicates untagged (endogenous) SHKBP1. FT, flow-through. (F) Representative live-cell images showing single particle tracking of endogenous mNG-p62 by confocal microscopy. HEK293T cell lines stably expressing mNG-p62 were transfected with either mScarlet-i (in both SHKBP1 KO and SHKBP1 WT backgrounds) or mScarlet-i-SHKBP1 (in SHKBP1 WT background, “OE”). Tracked p62 bodies are indicated by magenta circles, and movement tracks are shown by colored lines. Scale bars: 5 μm. (G) Quantification of the mean speed of tracked p62 bodies in SHKBP1 KO, WT, and OE conditions. n = 200–300 p62 bodies collected from three independently plated samples. Exact P values indicated from one-way ANOVA with Tukey’s post hoc test. (H) Representative live-cell images showing endogenous p62 body shapes. HEK293T stable cell lines were transfected with the indicated mScarlet-i constructs and observed by confocal microscopy 24 h after transfection. Scale bars: 5 μm. (I–L) Quantification of mNG-p62 body roundness, solidity, circularity, and aspect ratio. n = 1,000–1,300 p62 bodies collected from three independently plated samples. Exact P values indicated from one-way ANOVA with Tukey’s post hoc test. Source data are available for this figure: SourceData F5.