Fig 2: LY6E upregulated HIF-1a gene expression principally at the transcriptional levelA and B. HeLa (A) or MCF7 (B) cells were transiently transfected with either pcDNA4/LY6E (LY6E) or pcDNA4/myc-His A (EV) (A) or with either scramble-siRNA (Scr) or LY6E-siRNA (siLY6E-1, 2, or 3) (B), cultured under the indicated oxygen conditions, and subjected to western blotting for the indicated proteins. The intensity of the bands was quantified using ImageJ and relative HIF-1a expression levels (vs. ß-actin) are shown in the right panel (A). C. HeLa cells were transiently co-transfected with pG5H1bLuc, pRL-SV40, either pcDNA6/Gal4 or pcDNA6/Gal4DBD-HIF-1a TAD P564A, and either pcDNA4/LY6E or its empty vector, cultured under normoxic or hypoxic conditions for 24 h, and subjected to the luciferase assay. D and E. HeLa (D) or HeLa/ODD-Luc cells (E) were transiently transfected with either pcDNA4/LY6E or pcDNA4/myc-His A (EV) and with (E) or without (D) pRL-SV40, cultured under the indicated oxygen conditions in the presence (D) or absence (E) of 30 µM of the proteasome inhibitor, MG132, and harvested for Western blotting for the indicated proteins (D) or luciferase assay (E). F. HeLa/ODD-Luc cells were transfected with pRL-SV40 and either pcDNA4/LY6E (LY6E) or pcDNA4/myc-His A (EV), cultured under hypoxic conditions for 48 h, and treated with cyclohexamide (10 µg/mL) under normoxic conditions. After the reoxygenation treatment for the indicated period, cells were subjected to the luciferase assay. G–K. HeLa (G, H, and J) or MCF7 (I and K) cells were transiently transfected with pRL-SV40, either pcDNA4/LY6E or pcDNA4/myc-His A (EV), and either pGL3/HIF-1a 5'UTR-luc (G) or pHIF-1a promoter-luc (H and I), treated with (I and K) or without (G, H, and J) scramble-siRNA (Scr) or LY6E-siRNA (siLY6E-1, 2, or 3), cultured under either normoxic or hypoxic conditions (G-J) or under hypoxic conditions (K), and subjected to the luciferase assay (G-I) or qRT-PCR to quantify the mRNA levels of the indicated genes (J and K). L. HeLa/5HRE-Luc cells were transiently co-transfected with pRL-SV40 and either pcDNA4/LY6E (LY6E) or pcDNA4/myc-His A (EV), treated with indicated concentrations of Act-D, cultured under normoxic or hypoxic conditions, and subjected to the luciferase assay. Relative firefly luciferase activity to renilla luciferase activity in LY6E-expressing cells was further normalized by that in EV-transfected cells at each Act-D concentration. Mean ± s.d. n=4. *P < 0.05, **P < 0.001 (Student's t-test between the 2 indicated samples).

Fig 3: PTEN and PI3K/Akt signaling pathways were involved in the LY6E-mediated increase in HIF-1a transcriptionA-F. HeLa (A and C), HeLa/5HRE-Luc (B), or MCF7 (D-F) cells were transfected with either pcDNA4/LY6E (LY6E) or pcDNA4/myc-His A (EV) (A-C) either scramble-siRNA (Scr) or Akt-siRNA (C and D), or either scramble-siRNA (Scr) or LY6E-siRNA (siLY6E-1, 2, or 3) (E and F), and with (A, C, and D) or without (B, E, and F) pHIF-1a promoter-Luc, cultured under either normoxic or hypoxic conditions or under hypoxic conditions if not stated in the presence (A and B) or absence (C-F) of the indicated concentrations of LY294002, and subjected to the luciferase assay (A-D), qRT-PCR for PTEN mRNA levels (E), and Western blotting for the indicated proteins (F). G-J. HeLa cells were transfected with either pcDNA4/LY6E (LY6E) or pcDNA4/myc-His A (EV) and with (H-J) or without (G) either pcDNA4/PTEN-myc (PTEN) or pcDNA4/myc-His A (EV), cultured under normoxic or hypoxic conditions for 24 (G, H left, I, and J) or 48 (H right) hours, and subjected to qRT-PCR for PTEN mRNA levels (G and I), Western blotting for the indicated proteins (H), or the luciferase assay (J). Mean ± s.d. *P < 0.05, **P < 0.001 (Student's t-test between the 2 indicated samples).

Fig 4: LY6E upregulated HIF-1 activityA. HeLa/5HRE-Luc cells were transfected with pcDNA4/LY6E (LY6E) or pcDNA4/myc-His A (empty vector: EV) and pRL-SV40, cultured under normoxic or hypoxic conditions for the indicated period, and subjected to the luciferase assay. B. MCF7 cells transiently transfected with p5HRE-Luc were treated with scramble-siRNA (Scr) or LY6E-siRNA (siLYE-1, 2, or 3), cultured under normoxic or hypoxic conditions for the indicated period, and subjected to the luciferase assay. C. HeLa cells were transiently transfected with either pcDNA4/LY6E (LY6E) or pcDNA4/myc-His A (EV), cultured under normoxic or hypoxic conditions for 24 h, and then subjected to qRT-PCR to quantify the mRNA levels of the indicated genes. Mean ± s.d. n = 3. *P < 0.05, **P < 0.001 (Student's t-test between the 2 indicated samples).

Fig 5: Clinical implication of LY6E in human cancerA. PrognoScan database-based Kaplan-Meier analyses of the overall survival of 204 lung (data set: GSE31210; stage I-II lung adenocarcinomas) [46], 165 bladder (data set: GSE13507; histologically diagnosed transitional-cell carcinoma) [47], 74 brain (data set: GSE4412-GPL96; grade III and IV gliomas) [48], and 38 skin (data set: GSE19234; stage III and IV metastatic melanomas) [49] cancer patients stratified by high (red) and low (blue) LY6E expression levels (lung, high: n = 141, low: n = 63; bladder, high: n = 19, low: n = 146; brain, high: n = 17, low: n = 57; and skin, high: n = 8, low: n = 30). B. A BreastMark database-based Kaplan-Meier analysis of the overall survival of 2091 breast cancer patients stratified by high (blue) and low (red) LY6E expression levels. C. LY6E expression levels in basal-like breast cancer tissues (n=98) and normal breast tissues (n=22) from TCGA 2012 data. D. PrognoScan database-based Kaplan-Meier analyses of the overall survival of the same data set as A stratified by high (red) and low (blue) PTEN expression levels.