Fig 2: Differentiation of SH-SY5Y cells alters the expression of EML2 and EML4 splice variants, with the long variants showing stronger co-localization with microtubules compared to the short variants.A, the timeline of a method for differentiating SH-SY5Y cells. SH-SY5Y cells were cultured in a medium containing 10% FBS. After the cells reached 60% confluency (Day 0), the medium was replaced with fresh medium containing 1% FBS, and 10 μM Retinoic acid (RA) was added dropwise to cells. Red numbers indicate days on which cells were collected for Western blot analysis or fixed for immunostaining. Furthermore, the culture medium and RA were refreshed in the plates on the remaining days. B and C, lysates were prepared from SH-SY5Y human neuronal cells at the indicated days (D0 to D20) after the addition of RA to induce differentiation. Western Blots were undertaken with EML2, EML4 (N-term), and EML4 (C-term) antibodies. The α-tubulin antibody was used as a loading control. Molecular weights (kDa) are indicated on the left. Asterisks indicate quantified bands. Bar charts represent the intensity of EML2-L, EML2-S, EML4-L and EML4-S across the 20 days of induction with RA, normalized to α-tubulin. The EML4 bar chart on top shows the result with the EML4 N-terminal antibody, while the bottom ones show the result with the EML4 C-terminal antibody of both the long (EML4-L) and short (EML4-S) variants. Data obtained from three repeats, n = 3 (±S.D.). ∗∗∗ indicates a p-value of < 0.001, and ns (not significant) indicates a p-value of 0.1652. D and E, SH-SY5Y cells were fixed 24 h after transfection with EML2-L and -S variants (−RA) and 72 h after RA treatment following the transfection (+RA). Cells were stained with α-tubulin (red) and GFP (green) antibodies. DNA was stained with Hoechst 33258 (blue in merge); Magnified zoomed views are shown from the boxed areas. Scale = 10 μm. The mean Pearson's correlation coefficient for co-localization between EML2 variants and microtubules (MTs) in undifferentiated and differentiated SH-SY5Y cells was calculated from five measurements of 10 cells per experiment. n = 3 (±S.D.). p-value < 0.0001. Multiple coloured dots are the indication of different repeats. F and G, SH-SY5Y cells transfected with EML4-L and -S variants were fixed before (−RA) and after 72 h RA treatment (+R). Cells were then stained with α-tubulin (red) and GFP (green) antibodies. DNA was stained with Hoechst 33258 (blue in merge). Magnified zoomed views are shown from the box areas. Scale = 10 μm. The mean Pearson's correlation coefficient for co-localization between EML4 variants and microtubules (MTs) in undifferentiated and differentiated cells was calculated from five measurements per cell from three independent experiments. The experiment was repeated at least three times (±S.D.). ∗ p-value < 0.05.

Fig 3: Schematic representation of the potential splice variants of human EML2 and EML4.A, EML2 splice variants. The EML2 full length (variant 1) is made up of 22 exons and 850 amino acids (aa) consisting of a trimerization domain (TD) (orange) and a basic area (purple) in the N-terminus, and a TAPE domain (blue) in the C-terminus. EML2-Short (variant 2) lacks exons one to four and consists of 649 aa. Exon 1, which contains 57 aa, is absent from variant 3. Variant four lacks 317 aa from exons one through 7 as well as a portion of exon 8. Residue 172 is absent from variant 5 (red line), and exon one and residue 172 are absent from variant 6. Variant seven lacks 223 aa, exons one through five and a portion of exon 6. Variant CRAa contains 649 aa. This protein lacks 207 aa from the N-terminus, residue 234 is mismatched (red) and the residue 388 is missing. Variant CRAb contains the full portion of the N-terminus. In this protein residues between 234–388 and also 403–419 are mismatched and the rest of the C-terminus is missing. Accession numbers are indicated on the right. B, EML4 splice variants. EML4 full-length (variant 1) is comprised of 23 exons and 981 aa including a trimerization domain (TD) (orange) and a subsequent basic region (purple) in the N-terminus and the TAPE domain (blue) in the C-terminal region. EML4-Short (variant 2) is made up of 923 aa. 58 aa missed out from the N-terminus (red lines). An additional 11 aa are present in variant three between residues 222 and 223 (green). Variant X1 is the combination of variants 2 and 3. The N-terminus lacks 58 aa, while there are an extra 11 aa between residues 222 and 223. In variant X2 the first 79 aa from N-terminal are missing. There are an extra 11 aa between residues 222 and 223 and also residues 282 and 382 are mismatched (red). Variant X3 contains an extra 11 aa between residues 222 and 223 and also residues 282 and 382 are mismatched. Accession numbers are indicated on the right.

Fig 4: Summary of the impact of EML2 and EML4 splice variants overexpression and depletion on SH-SY5Y neurite length and branching.A, overexpression of EML2-L with or without RA increases the length of neurite and decreases branching while the overexpression of EML2-S decreases the length of neurite and increases branching. Overexpression of both EML4-L and -S does not alter cell morphology with or in the absence of RA. The addition of RA increases the overall length of neurites. B, depletion of either EML2 or EML4 increases the length of neurite and branching. C, neurite extension happens when EML2-L stabilizes small microtubules in the neurite shaft prior to their entry into the dendritic spine or growth cone. As a result, the membrane is propelled forward, producing longer microtubules that in turn form longer neurites. Meanwhile, EML2-S promotes the formation of numerous short microtubules that can push the membrane in various directions leading to an increase in neurite branching.