Figure S2.

ALAL-1 promotes the oncogenic phenotype of different lung cancer cells. (A) Diagram showing the location of the sgRNAs used for deleting a region of 500 bp flanking exon 3 of ALAL-1. The annealing sites for the primers used (forward [fwd]; reverse [rev]) to screen for the deletion are indicated. BLAT results of the obtained sequences of the PCR products show the flanking sequences of the deletion. (B) DNA electrophoresis showing the PCR products obtained with the fwd/rev in and out primers using genomic DNA from the parental HCC95 cells and clones (23 and 24). (C) Crystal violet absorbance measuring the colony formation capacity of the CRISPR/Cas9 engineered cells. (D) Percentage of apoptotic cells from the CRISPR/Cas9 clones in which ALAL-1 expression was reduced. (E) Cell proliferation measured by MTS of HCC95 cells after ALAL-1 inhibition. (F) Crystal violet (CV) absorbance measuring the colony formation capacity in HCC95 cells transfected with siRNAs (1 and 2) targeting ALAL-1 or control siRNA. (G) Percentage of HCC95 apoptotic cells measured by flow cytometry after ALAL-1 inhibition. (H) Inhibition levels of ALAL-1 obtained with siRNAs 1 and 2 in H1648 cells. RNA levels are normalized to HPRT and relative to the control siRNA. (I) Cell proliferation measured by MTS of H1648 cells after ALAL-1 inhibition. (J) Crystal violet absorbance measuring the colony formation capacity in H1648 cells transfected with siRNAs (1 and 2) targeting ALAL-1 or control siRNA. Absorbance values are represented relative to the control. (K) ALAL-1 levels in HCC95 cells transiently transfected with the pcDNA3–ALAL-1 plasmid. (L and M) Clonogenic assay of HCC95 cells transiently overexpressing ALAL-1. (N) ALAL-1 levels in H1648 cells transfected with the pcDNA3–ALAL-1 plasmid. (O and P) Clonogenic assay of H1648 cells transiently overexpressing ALAL-1. (Q) Overexpression levels of ALAL-1 in A549 cells transduced with a retrovirus expressing the lncRNA. (R and S) Colony formation assay of A549 cells overexpressing ALAL-1. (T) Volumes of tumors formed by subcutaneous injection of wt, stably expressing empty vector, or ALAL-1–overexpressing A549 cells in immunocompromised mice. Boxplot of n = 7; significance was determined by one-tailed Mann-Whitney test. All other significances were determined by two-tailed unpaired t test and represented as *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. ctrl, control; siALAL-1, ALAL-1 siRNA; siCtrl, control siRNA. Error bars represent SEM. In T, 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.