Fig 2: Ciliary localization of MAST4R503A and MAST4D504A.(A, B, C, D) Representative confocal images of serum-starved RPE-1 cells transfected with the indicated mutant MAST4-mCherry (mC) (red) and stained for Ac-Tub (green) and γ-Tub (cyan). (A, C) Low-power views show a single transfected cell. The high-power view highlights the ciliary distribution of MAST4-mCherry. (B, D) Profile blots of fluorescence intensities (FIs) are also shown. BB, basal body. White dashed lines in the low-power view demarcate the cell border (DAPI: nuclear). Scale bars: 10 μm (low-power magnification) and 1 μm (high-power magnification). The y-axis FI unit is defined by the acquired values divided by 104 using Zen software (Zeiss). The x-axis describes the distance from the proximal part of the basal body (considered as 0). The representative line scans from the images of 11–16 cilia are shown.

Fig 3: Validation of the KD effect and overexpressed MAST4.(A) Representative immunoblots of endogenous MAST4 and GAPDH in RPE-1 cells transfected with GFP alone (control), MAST4-shRNA1-IRES-GFP (MAST4-sh1), or MAST4-shRNA2-IRES-GFP (MAST4-sh2). (B) Lysates of RPE-1 cells transfected with GFP alone (control), or GFP together with MAST4WT were immunoblotted with anti-MAST4 or anti-GAPDH antibody. The high level of transfected MAST4 obscured the relatively weaker signal of the endogenous MAST4 signal.

Fig 4: Mapping catalytic motif residues of MAST4 required for the ciliary base activation of phospho-(T94)Tctex-1.(A) Alignment of the kinase domain of MAST2 and PKA catalytic subunit α (PKACA) with the predicted catalytic domain of MAST4. Bold letters were used to highlight the amino acid residues that were changed to alanine by site-directed mutagenesis. The predicted catalytic motif (H502–N509), DFG motif (D522–G524), and autophosphorylation residues (T524 or T535) of MAST4 are marked by a bar. (B) Ciliary resorption assay of RPE-1 cells transfected with GFP alone (control), or GFP together with MAST4WT or the indicated MAST variants. The histogram shows the percentage of ciliated GFP+ cells at different time points after serum readdition. Values are means ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. n = 100 cells in each experiment, with three independent experiments. (C) Representative confocal images of phospho-(T94)Tctex-1 (magenta) and Ac-Tub (green) in RPE-1 cells transfected with GFP together with WT or indicated mutant MAST4-mCherry under the starving (0 h) and 2-h serum-stimulated conditions. (C) Phospho-(T94)Tctex-1 was detected by Alexa Fluor 647–conjugated secondary antibody and is pseudo-colored in magenta for presentation. n = 12–16 cells from three independent experiments. (D) Fluorescence intensity of phospho-(T94)Tctex-1 associated with the proximal end of Ac-Tub–labeled cilium was quantified (D). Values are means ± S.E.M. ##P < 0.01 and ###P < 0.001, two-way ANOVA followed by Bonferroni’s test. §§§P < 0.001, t test. n = 12–16. Scale bar: 1 μm (C).

Fig 5: MAST4 is localized in the cilium and required for ciliary resorption.(A) RPE-1 cells transfected with the GFP alone (control), MAST4-shRNA1-IRES-GFP (MAST4-sh1), or MAST4-shRNA2-IRES-GFP (MAST4-sh2) were starved and added serum back for the indicated time periods. Representative confocal images of the Ac-Tub–labeled cilium (red) and the γ-Tub–labeled basal body (cyan) in GFP+ cells (green) are shown. Cell nuclei were stained with DAPI (blue). n = 16–31 cells from three independent experiments. Scale bar: 5 μm. (B, C) Quantification of (A). Histogram shows the cilium length (measured by Ac-Tub) (B) and the fraction of the GFP+ cells expressing a cilium (C). Values are means ± S.E.M. *P < 0.05 and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. #P < 0.05 and ##P < 0.01, two-way ANOVA followed by Bonferroni’s test. n = 16–31 cells (B) and n = 100 cells in each experiment, with three independent experiments (C). (D) RPE-1 cells transiently transfected with GFP (control) or GFP together with MAST4WT were starved and followed by serum readdition. The quantification of the percentage of GFP+ cells, harvested at different time points post-serum stimulation, is shown. Values are means ± S.E.M. *P < 0.05 and ***P < 0.001, one-way ANOVA followed by Tukey’s test versus 0 h for each group. n = 100 cells in each experiment, with three independent experiments. (E) Representative confocal images of MAST4-mCherry (mC) in quiescent transfected RPE-1 cells stained for mCherry (red), Ac-Tub (green), and γ-Tub (cyan). White dashed lines demarcate the cell border (DAPI: nuclear). The high-power view highlights the ciliary distribution of MAST4-mCherry. Scale bars: 10 μm (E, low-power magnification) and 1 μm (E, high-power magnification). (E, F) Segmented profiles of fluorescence intensities (FIs) along the white dashed arrow shown in (E). BB, basal body. The y-axis FI unit is defined by the acquired values divided by 104 using Zen software (Zeiss). The x-axis describes the distance from the proximal part of the basal body (considered as 0). The representative line scan from the images of 16 cilia is shown.