Fig 2: SG2NA is regulated at protein level by proteasomal degradation and its hyperphosphorylated form is more stable. NIH3T3 cells were treated with (A) 100 μM Okadoic acid alone and 10 μM MG132 and mixture of both (C) 10 μM MG132 alone (E) 10 mM LiCl (H) 25 μM PD98059, for various time points and western analysis was done with SG2NA and β-actin antibody. Graph representing quantitaive analysis of relative expression of SG2NA in time dependent manner upon their treatment is shown in (B), (D), (F) and (I) repectively. Confocal images of the cells treated with LiCl and PD98059 is shown in (G) and (J) respectively. Magnification- 60X, scale bar- 10 μm. (K) NIH3T3 cells were treated with various inhibitors as LiCl, PD98059 and LY294002 for 12 hrs and cell lysates were immunoprecipitated with SG2NA antibody followed by blotting with phosphoserine, phosphothreonine and SG2NA antibodies. Equal loading is shown by β-actin. (L) Graphs representing relative level of respective proteins with β-actin as loading control is shown. Error bars are the standard deviation of 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Full length blots for panel (A), (C), (E), (H) and (K) are shown in Supplementary Figure S1.

Fig 3: NIH3T3 has only 78 kDa isoform enriched in cytosol and microsomes. (A) NIH3T3 cells were harvested and 75 µg of protein lysates were loaded on SDS-polyacrylamide gel and blotted with SG2NA antibody. Protein was extracted from transiently transfected HEK293T cells harbouring various isoforms of SG2NA i.e., 35 kDa, 52 kDa, 78 kDa and 87 kDa were loaded to act as markers. (B) NIH3T3 cells were fractionated and each organelle rich fraction was loaded and blotted with SG2NA antibody, authenticity of the fractions were confirmed by probing with organelle specific markers. Full length blots for panel (B) are shown in Supplementary Figure S1.

Fig 4: SG2NA downregulation increases the G1 phase duration while upregulation increases G2 phase duration. (A) Cells stably overexpressing 78 kDa and (B) underexpressing SG2NA were harvested, 100 µg of cell lysates were loaded on SDS-polyacrylamide gel and immunoblotted with SG2NA and ß-actin antibody. Numbers on the top of bands represent fold change in protein levels relative to untransfected NIH3T3 cells. (C) Representative FACS histograms of NIH3T3 cells stably transfected with vector alone, 78 kDa SG2NA and shRNA against SG2NA. (D) Graphical representation of the percentage of cells present in different phases of cell cycle during the cell cycle preogression. (E) Left panel: Lysates of normal NIH3T3 cells and SG2NA downregulated cells were loaded on SDS-polyacrylamide gel and probed with cyclin D1 and ß-actin. Right panel: Graph representing real time analysis by qPCR for cyclin D1 mRNA expression in normal NIH3T3 cells and SG2NA downregulated cells. Error bars are the standard deviation of 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Full length blots for panel (A) and (B) are shown in Supplementary Figure S1.

Fig 5: 35 kDa variant of SG2NA has no role in cell cycle. (A) Cells stably overexpressing 35 kDa were harvested, 100 µg of total protein lysate was loaded on SDS-polyacrylamide gel and probed with SG2NA and ß-actin. (B) 35 kDa overexpressing NIH3T3 cells were fractionated and organelle rich fractions loaded on SDS-polyacrlamide gel followed by western analysis using SG2NA antibody. (C) Representative FACS histograms of NIH3T3 cells stably transfected with 35 kDa SG2NA. (D) Graphical representation of the percentage of cells present in different phases of cell cycle during the cell cycle preogression. Error bars are the standard deviation of 3 independent experiments. (E) Schematic representation of proposed process. SG2NA remains in hyperphosphorylated state in the presence of okadoic acid. This hyperphosphorylated SG2NA is more stable. PD98059 inhibits phosphorylation of MAPK kinase and thus ERK. The decrease in phosphorylation of ERK results in decreased activity of ERK thereby decreasing inhibitory phosphorylation of GSK3ß which inturn increases pSG2NA level thus increases its stability. LiCl is known to inhibit GSK3ß activity by increasing its Ser-9 phosphorylation, thereby not phosphorylating SG2NA hence decreased level of SG2NA was observed. Full length blots for panel (A) are shown in Supplementary Figure S1.