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. 2022 Nov 1;41(5):111580. doi: 10.1016/j.celrep.2022.111580

Figure 4.

Figure 4

Targeting upstream FA regulator PTEN rescues FA and vimentin bundling phenotypes in sacsin KO cells

(A) Regulators and effectors of FA signaling.

(B) Western blots for regulators FAK, pFAK-Tyr397, total and phosphorylated JNK, total and phosphorylated PAX, phosphorylated JUN, and PTEN in total cell lysates from sacsin KO and control cells.

(C) Confocal images of cells immunolabeled for pFAK. Scale bar, 10 μm.

(D) Western blots of WT/KO cells treated with siRNAs targeting PTEN or scrambled.

(E–G) Quantification of PTEN (E), pFAK (F), and pPAX (G) levels in WT/KO cells treated with scrambled or PTEN targeting siRNAs. n = 3, SEM, Student’s t test, ∗p < 0.05, ∗∗∗p < 0.001.

(H) Confocal images for cells transfected with siRNAs targeting PTEN or scrambled, and immunolabeled for vimentin and vinculin. Arrowheads indicate cells with prominent FAs, arrows indicate cells with absent or reduced perinuclear accumulations of vimentin. Scale bars, 10 μm.

(I and J) Quantification of the incidence of sacsin KO cells with perinuclear accumulations of vimentin (I) or vinculin-positive FAs (J) 48 h after transfection with siRNAs targeting PTEN or scrambled siRNAs. n = 3 replicates, >100 cells in each replicate, SEM, Student’s t test, ∗p < 0.05, ∗∗∗∗p < 0.0001.