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. 2020 Aug 27;219(9):e201908078. doi: 10.1083/jcb.201908078

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© 2020 Athie et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S5. — ALAL-1 acts with SART3. (A) View of the genomic region of ALAL-1 with mapping of the qRT-PCR primers used to detect ALAL-1. (B) ALAL-1 levels in HCC95 WT and ALAL-1 CRISPR depleted cells (clone 23) detected using different primer sets, as shown in A. (C) ALAL-1 exon sequences (exons 1–3) with SART3 putative binding sites marked in red. (D) Clonogenicity assay of HCC95 cells transfected with ALAL-1 (full length and fragments described in Fig. 5 D) and depleted or not depleted of SART3 by RNAi. (E) Clonogenicity assay of HCC95 cells depleted or not depleted of ALAL-1 by CRISPR and transfected with an overexpressing ALAL-1 vector. (F) ALAL-1 levels in cells used in D. (G) Clonogenicity assay of A549 cells stably overexpressing ALAL-1 or not (empty) and depleted of SART3 by RNAi or not (siRNA-Ctrl). (H and I) ALAL-1 and SART3 RNA levels in cells used in F. (J) SART3 localization in A549, HCC95, and H1648 cells identified by Western blot (anti–SART3 ab). Lamin A/C and GAPDH were used as controls of nuclear and cytoplasmic fractions, respectively. (K) Effect of ALAL-1 inhibition on SART3 mRNA and protein levels. (L) ALAL-1 and SART3 mRNA levels quantified by qRT-PCR in the samples used for RNA-seq analyses. (M) Number of differentially expressed genes (adj. P < 0.05) determined by the RNA-seq analysis in the indicated conditions. (N) Graphs of colocalization quantifications of SART3/ALAL-1 and USP4/ALAL-1 (shown in Fig. 6) normalized to ALAL-1 signal. Graphs of mean (± SEM) for three independent experiments are shown. Significance was determined by two-tailed unpaired t test and represented as **, P ≤ 0.01. Ctrl, control; ns, not significant; siALAL-1, ALAL-1 siRNA; siCtrl, control siRNA; OE, overexpression; Abs, absorbance; ab, antibody; Nuc, nucleus; Cyt, cytoplasm; siSART3, SART3-targeting siRNA. Bottom and top of the box are the 25th and 75th percentile (the lower Q1 and upper quartiles, respectively Q3), and the band near the middle of the box corresponds to the median. The lower whisker extends Q1 – 1.5 * interquartile range (IQR) and the upper one Q3 + 1.5 IQR.