Fig 2: Ingenuity Pathway Analysis (IPA) of Proteins Expressed in SP Cells.(A) Localization; (B) Biological processes of identified proteins. We categorized the proteins based on their functional assignments using Ingenuity Pathway Analysis. The molecular functions reported include cell death, metabolism, cellular organization, metabolism of DNA, protein degradation, processing of RNA, production of reactive oxygen species, nitric oxide, molecular transport, cell cycle, folding protein, and cellular movement. % = 100 X number of identified proteins/all 932 proteins analyzed. (C), A Venn diagram confer to Table 2. Forty proteins were significantly increased in OCUM-12/SP cells compared to their parent OCUM-12 cells. Thirty-five proteins were significantly increased in OCUM-2MD3/SP cells compared to their parent OCUM-2MD3cells. Eight candidate proteins, RBBP6, HSPA4, HSPA9, GLG1, DCTPP1, VPS13A, CK18 and ALDOA, overlap in both OCUM-12/SP and OCUM-2MD3/SP cells. (D), mRNA expression. RT-PCR analysis indicated that the expression level of RBBP6, HSPA4, DCTPP1, HSPA9, VPS13A, ALDOA, GLG1, and CK18 was high in OCUM-12/SP (9.15 fold, 9.36 fold, 4.14 fold, 7.80 fold, 2.08 fold, 1.46 fold, 3.44 fold, and 1.99 fold, respectively) and OCUM-2MD3/SP (6.15 fold, 1.71 fold, 2.33 fold, 2.30 fold, 2.03 fold, 1.32 fold, 1.35 fold, and 1.31 fold, respectively), in compared with the control of parent OCUM-12 and OCUM-2MD3. (E), Correlation of Signaling Pathways between RBBP6 and Differentially-Expressed Proteins in CSC-like SP cells. RBBP6 is over-expressed (red) in CSC-like SP cells (OCUM-12/SP cells and OCUM-2MD3/SP cells). Hsp90, HSPA4, and TAGLN2 (pink) up-regulated in CSC-like SP cells were associated with the RBBP6 signaling pathway.

Fig 3: In situ hybridisation of candidate genes. (a) PAX6 protein (green) at 12pcw. a-a’*) show high magnification images of PAX6 protein expression in the (a’) SP/CP, a*) IZ, a”) SVZ and a’*) VZ. Low magnification scale bars = 50 µm, high magnification scale bars = 10 µm. b) Low magnification image of KIF22 mRNA in the 12pcw human fetal cortex, b’) High magnification showing KIF22 mRNA (blue) to be predominantly expressed in the germinative zones. c) Low magnification image of ALDOA mRNA in the 12pcw human fetal cortex, (b’) High magnification showing ALDOA mRNA (blue) to be predominantly expressed in the germinative zones but also some expression in the IZ and CP. (d) Low magnification image of HIRIP3 mRNA in the 12pcw human fetal cortex, (d’) High magnification showing HIRIP3 mRNA (blue) to be expressed throughout the telencephalic wall. e) Low magnification image of PAGR1 mRNA in the 12pcw human fetal cortex, e’) High magnification showing PAGR1 mRNA (blue) to be expressed throughout the telencephalic wall. (f) Low magnification image of MAZ mRNA in the 12pcw human fetal cortex, (f’) High magnification showing MAZ mRNA (blue) to be expressed throughout the telencephalic wall. For ISH, low magnification scale bars = 2 mm and high magnification scale bars = 100 µm.

Fig 4: Effects of siRNA-mediated knockdown of expression of AldoA, EWSR1 and ILF3-90 on enterovirus replication.A, B. HeLa cells were transfected with siRNAs specific to AldoA, EWSR1 and 90KDa isoform of ILF3, or non-targeting control siRNA, and polio or Coxsakie B3 replicon replication assays were performed 72 h post siRNA transfection. The total replication signal was calculated as the area under the corresponding kinetics curves. Cell viability signal is proportional to the level of ATP in cells. Western blots show the efficacy of siRNA-mediated knockdown of the targeted proteins. C. HeLa cells were transfected with siRNAs specific to AldoA, EWSR1 and 90KDa isoform of ILF3, or non-targeting control siRNA. 72 h post siRNA transfection cells were infected with an MOI of 1 PFU/cell of poliovirus or Coxsackie virus B3, and the total virus yield was determined at 6 h p. i. Western blots show the efficacy of siRNA-mediated knockdown of the targeted proteins. D. HeLa cells were transfected with siRNAs specific to 90KDa isoform of ILF3 or EWSR1 or a non-targeting control siRNA. 72h post siRNA transfection, the cells were transfected with a replication-defective replicon RNA containing the ?3D mutation. The total translation signal was calculated as the area ander the curve from 1 to 4 h post ?3D RNA transfection. Western blots show the efficacy of siRNA-mediated knockdown of the targeted proteins. E. HeLa cells were transfected with siRNAs specific to AldoA, EWSR1 and 90KDa isoform of ILF3, or non-targeting control siRNA, and cell viability assays detecting the level of ATP or the activity of the mitochondrial respiratory chain enzymes were performed 72h post siRNA transfection. Western blots show the efficacy of siRNA-mediated knockdown of the targeted proteins.

Fig 5: High expression of ALDOA is highly expressed in LSCC metastasis.(A) Tissue microarray (TMA) analysis of ALDOA expression in 75 pairs of matched LSCCs and adjacent normal tissues. IHC staining assays were performed to examine ALDOA protein expression and three pairs were shown as representatives. (B) In normal tissues 22 cases (29.3%) were measured as positive (+), and 1 case (1.3%) was strong positive (++). In LSCC tissues 25 cases (33.3%) were measured as positive (+) and 25 cases (33.3%) were strong positive (++).