Figure 2.

Fibroblasts Derived from an Additional TBS Individual Exhibit Cilia Anomalies

(A) Clinical findings in individual TBS^p.Arg276∗ (TBS²⁷⁶).

(B) Western blot analysis of lysates from control ESCTRL#2 and TBS²⁷⁶. Samples were run in duplicate on the same gel and probed against SALL1^FL (asterisk, R&D antibody) or against SALL1²⁷⁶ (black arrowhead, N-terminal specific antibody⁶) TBS²⁷⁶ cells encoded with a ∼48 kDa truncated protein that positively reacts against SALL1 antibody. Vinculin and GAPDH were used as loading controls. Molecular-weight markers are shown to the right.

(C and D) Confocal micrographs showing endogenous SALL1^FL localization in control ESCTRL#2 (C) or TBS²⁷⁶ (D) fibroblasts detected by FL-specific antibody (R&D). DAPI was used to label the nuclei (blue), and black and white images show the single green channel. Scale bars, 5 μm.

(E and F) Immunofluorescence micrographs showing cilia marked with acetylated tubulin (purple), basal bodies marked with ODF2 (green), and nuclei marked with DAPI (blue) in control ESCTRL#2 and TBS²⁷⁶ fibroblasts. Pictures were taken with a Zeiss Axioimager D1 fluorescence microscope with a 63× objective. Scale bars, 2.5 μm.

(G and H) Graphical representation of cilia length measurements (G; n = 123 and 235 cilia for control ESCTRL#2 and TBS²⁷⁶ fibroblasts, respectively) and cilia frequency (H; n = 28 and 31 micrographs for control ESCTRL#2 and TBS²⁷⁶ fibroblasts, respectively) of micrographs as shown in (E) and (F). Three independent experiments were pooled together. The graphs represent the mean and SEM. The p values were calculated with a two-tailed unpaired Student’s t test: ^∗∗∗p < 0.001.

(I and J) Confocal micrographs showing SALL1^FL-YFP localization in control ESCTRL#2 and TBS²⁷⁶ fibroblasts. Actin is labeled by phalloidin (purple), and nuclei are labeled by DAPI (blue). Black and white images show the single green channel. Scale bars, 5 μm.