SUGP1 sustains functional levels of LIS1 mRNA and protein. (A) Schematic of SUGP1 domain structure. NLS, nuclear localization signal. (B) Representative images of SUGP1 intensity and GFP-BIC2N-FRB and PTS-RFP-FKBP localization in U-2 OS PEX cells treated with crSUGP1 #1. Scale bar, 25 µm. (C) Scatter plot of mRNA abundance for crSUGP1 #1-edited versus NTC-treated U-2 OS cells (mean log₂ normalized values from three independently performed experiments). mRNAs meeting the threshold for inclusion (minimum absolute log₂ fold change ≥0.5; FDR ≤ 0.05) are labeled in blue, except SUGP1, DYNC1I2, and LIS1, which are labeled in yellow. The inset table shows non-logarithmic values for SUGP1, DYNC1I2, and LIS1. See Table S9 for full results. (D) Quantification of endogenous DYNC1I2 and LIS1 protein signal (determined by immunofluorescence) in unmodified U-2 OS cells treated with NTC or crSUGP1 #1 or #2 and transfected with a control (iRFP670) or crRNA-resistant SUGP1-V5 expression plasmid. (E) Representative images and quantification (perinuclear versus peripheral localization ratio) of GFP-BICD2N-FRB and PTS-RFP-FKBP localization in U-2 OS PEX cells treated with NTC or crSUGP1 #1 or #2 and transfected with a control (iRFP670), crRNA-resistant SUGP1-V5, or LIS1-FLAG expression plasmid. Scale bar, 25 µm. In D and E, cells were transfected with crRNA 96 h before fixation, and with expression plasmid 48 h after crRNA transfection. Data points represent mean per cell intensity values (D) or mean per cell localization ratio values (E) aggregated at well level from four independent experiments (minimum of 100 transfected cells analyzed per well; four wells analyzed per condition). Error bars signify SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA with Tukey’s multiple comparison; colors of asterisks indicate comparison group).