Fig. 2: Deletion of TSSK1B reduces YAP phosphorylation and elevates YAP activity.

A HEK293A, A375P, and U2OS cells were treated with shCon and shTSSK1B and qRT-PCR was performed using specific primer pairs to quantify the mRNA levels of TSSK1B, CTGF, and CYR61. The measurements were normalized with respect to the HPRT1 mRNA levels. Error bars depict mean ± SEM (n = 3). ***p < 0.001. **p < 0.01. *p < 0.05. Student’s t-test was used for statistical analysis. B HEK293A, A375P, and U2OS cells were transduced using shCon or shTSSK1B, and knockdown efficiency for TSSK1B was confirmed by immunoblotting. Vin: Vinculin. C HEK293A cells treated with shCon or shTSSK1B were subjected to immunofluorescence staining for TSSK1Β (green) and YAP (red); 4’,6-diamidino-2-phenylindole (DAPI) (blue) was used for nuclear staining. Scale bars, 10 µm. The fluorescence intensity of TSSK1B, YAP, and DAPI was quantified by drawing a line (yellow) of 25 μm in a merged figure using the ZEN 3.5 blue edition program. D Knockout of TSSK1B using CRISPR/Cas9 in U2OS cells was confirmed by immunocytochemistry. Scale bars, 10 µm. The fluorescence intensity of TSSK1B, YAP, and DAPI was quantified by drawing a line (yellow) of 25 μm in a merged figure using ZEN 3.5 blue edition program. E HEK293A cells treated with shCon and shTSSK1B were incubated with serum-free medium (S.S) for 2 h, 25 mM 2-Deoxy glucose (2-DG) for 2 h, 1 mM metformin (Met) for 4 h, or 0.25 μg·ml⁻¹ latrunculin B (LatB) for 30 min, and whole cell lysates were immunoblotted. PT: phos-tag gel. F U2OS WT and TSSK1B KO cells were incubated with serum-free medium (S.S) for 2 h, 25 mM 2-Deoxy glucose (2-DG) for 2 h, 1 mM metformin (Met) for 4 h, or 0.25 μg·ml⁻¹ latrunculin B (LatB) for 30 min. Whole-cell lysates were immunoblotted. PT: phos-tag gel. G shCon- or shTSSK1B-treated HEK293A cells were incubated with 1 mM metformin for 4 h. The mRNA levels of CTGF and CYR61 were determined. The relative unit values for mRNA were normalized to one with respect to that of the control group without metformin treatment. Error bars depict mean ± SEM (n = 3). ***p < 0.001. *p < 0.05. NS: Not significant. Student’s t-test was used for statistical analysis.