Fig 2: Relative gene expression and distribution of human SCDs in different tissues. Relative mRNA expression of SCD5A (A), SCD5B (B), SCD1 (C) and cumulated relative SCD gene expression (D) were measured in ten different human tissues by qPCR, as described in Section 4. GAPDH gene expression served as a control. The distribution of SCD5 transcript variants (E) and all three SCD mRNAs (F) in human tissues is represented on a percentage scale, where total SCD5 or total SCD expression is considered as 100 percent. Experiments were performed in triplicate. In charts (A–C), data are shown as the mean values ± S.D. The S.D. values for sections (D), (E) and (F) are presented in Tables S2, S3 and S4, respectively.

Fig 3: Effect of natural sequence variations predicted by the NetGene2-2.42 program on the recognition probability of SCD5A- and SCD5B-specific donor and acceptor sites. (A) Position of SNVs affecting donor and acceptor sites critical for alternative splicing in the SCD5 gene. Exons are numbered and marked with rectangles. SCD5A- and SCD5B-specific, as well as shared exons are shown in green, red and gray, respectively. In addition to localization, nucleotide exchanges caused by SNVs are also indicated. (B) Probabilities of donor and acceptor sites for SCD5 sequence variants predicted by NetGene2-2.42, as indicated in Section 4. Probabilities are plotted as a function of sequence variations at a given donor or acceptor site. The black, blue, green and red columns in the diagram refer to wild type, AB donor site SNV, B acceptor site variants and A acceptor site variants, respectively.

Fig 4: Minigene analysis of unequal SCD5 alternative splicing in HEK293T cells. (A) Schematic structure of SCD5 minigene construct. Exons are numbered and marked with rectangles. The length of the segments cloned from 5′ and 3′ regions of introns 3 and 4 are also shown. The stop codons and restriction endonuclease cleavage sites are indicated. RT-PCR primers used to verify A- and B-specific splicing events are indicated by arrows. SCD5A- and SCD5B-specific and shared primers are shown in green, red and black, respectively. (B) Verification of AS in HEK293T cells transfected with the SCD5 minigene plasmid. The RT-PCR products specific for SCD5 transcript variants were separated and visualized on 2% agarose gel. GAPDH served as an endogenous control. Representative results of four independent experiments are shown. (C) The distribution of endogenous and SCD5 minigene-derived SCD5A and SCD5B expression was measured by qPCR and is represented on a percentage scale. Experiments were performed in triplicate. The S.D. values are shown in Table S6. (D) SCD5A and SCD5B protein levels were detected by immunoblotting in samples prepared from pcDNA3.1(−) and SCD5 minigene transfected cells. HEK293T cells were harvested and processed 24 h after transfection. Aliquots of cell lysates (5 µg) were loaded on 15% SDS-polyacrylamide gel, transferred to Immobilon-P membrane, and detected by SCD5-specific antibody. Actin was measured as a loading control. Representative immunoblots of six independent experiments are shown. (E) The band intensities of immunoblots were quantified by densitometry and SCD5 protein distribution is represented on a percentage scale. The S.D. values are shown in Table S6.

Fig 5: Transcript variants of human SCD5 gene. Schematic gene (A), mRNA (B) and protein (C) structure of human SCD5A and B transcript variants. Exons are numbered and marked with rectangles. Introns are depicted with straight lines and UTRs with diagonally lined rectangles. The start and stop codons, and the length of introns and exons are also indicated. SCD5A- and SCD5B-specific and shared exons are shown in green, red and gray, respectively. The position of the putative TM domains and His-boxes in the protein are also marked. Verification of SCD5A- and SCD5B-specific splicing events by RT-PCR (D) and direct sequencing (E). RT-PCR primers used to verify A- and B-specific splicing events are indicated by arrows, SCD5A- and SCD5B-specific and shared primers are shown in green, red and black, respectively. The agarose gel images represent typical results of three independent experiments. The electropherograms show the AS hotspot of the two transcript variants indicating the starting point of the different 3′ ends. (aa: amino acid; nt: nucleotide; TM helix: transmembrane helix; His-box: histidine containing sequence).