Journal: Journal of Hematology & Oncology

Article Title: A cytoplasmic long noncoding RNA LINC00470 as a new AKT activator to mediate glioblastoma cell autophagy

doi: 10.1186/s13045-018-0619-z

Figure Lengend Snippet: FUS interacted with LINC00470 and AKT to form a ternary complex in the cytoplasm. a The interaction of LINC00470 and FUS was detected through RIP assays in U251 cells. Data are presented as the mean ± S.E.M. of three independent experiments. ** p < 0.01. b RNA pulldown showed binding between LINC00470 and FUS. c RIP assays showed that there was no interaction between LINC00470 and AKT in U251 cells. Data are presented as the mean ± S.E.M. of three independent experiments. d HEK293 cells were transfected with HA-AKT, Flag-FUS, and pcDNA3.1-LINC00470. Two-step co-immunoprecipitation verified their interaction. The expression levels of LINC00470, AKT, and FUS were measured with RT-qPCR and Western blotting, respectively. e The localization of AKT and FUS was detected by immunofluorescence staining in HEK293 cells. f The co-localization of AKT and FUS was detected by immunofluorescence staining in U251 cells. g Left, the interactions between endogenous FUS and AKT in the cytoplasm and nucleus were measured by co-immunoprecipitation; right, an RNA pulldown assay showed binding between endogenous LINC00470 and AKT in the cytoplasm and nucleus of U251 cells transfected by si-FUS. h Western blotting detected the expression of FUS in GBM cells transfected by si-FUS. Expression levels of AKT and p-AKT S473 were measured by Western blotting in GBM cells that re-expressed LINC00470 in FUS-KD GBM cells. i Western blotting detected the expression levels of p-AKT S473 in the cytoplasm and nucleus of U251 cells transfected by si-LINC00470

Article Snippet: The following primary antibodies were used: AKT (rabbit, Proteintech, 10176-2-AP, WB1:1500, IP:1:250, RIP:1:100); FUS (rabbit, Abcam, ab23439, WB1:2000, IP1: 200, RIP1:100); phospho-Akt (Ser473) (rabbit, Cell Signaling, #4060, WB1:1500); phospho-Akt (Thr308) (rabbit, Cell Signaling, #13038, WB1:1500); hexokinase I (rabbit, Cell Signaling, #2024, WB1:1000); hexokinase II (rabbit, Cell Signaling, #2867, WB1:1000); Flag (mouse, Sigma-Aldrich, F1804, IP 1:200); GAPDH (mouse, Sangon, D190090, WB 1:5000); H3 (rabbit, Beyotime, AH433, WB 1:500); and p53 (mouse, Active Motif, 39739, WB 1:1000, RIP 1:150).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Journal of Hematology & Oncology

Article Title: A cytoplasmic long noncoding RNA LINC00470 as a new AKT activator to mediate glioblastoma cell autophagy

doi: 10.1186/s13045-018-0619-z

Figure Lengend Snippet: FUS bound to AKT and promoted AKT activation. a Co-IP analysis measured the exogenous interaction between FUS and AKT in HEK293 cells. b Representative immunofluorescence staining displayed the co-localization of FUS and AKT in the nucleus of HEK293 cells. c Co-IP analysis measured the endogenous interaction between FUS and AKT in U251 cells. d Representative immunofluorescence staining displayed the endogenous co-localization of FUS and AKT in the cytoplasm of U251 cells. e Left, GST pulldown assays showed that the GGR domain of FUS pulled down AKT; right, GST pulldown assays showed that the N-terminal region of AKT mainly pulled down FUS. f Upper, Western blotting measured the expression levels of FUS and AKT in GBM cells transfected by si-FUS; lower, Western blotting measured the expression levels of AKT and FUS in GBM cells transfected with si-AKT. g Western blotting measured the expression levels of AKT and pAKT in the whole lysis, cytoplasm, and nucleus of U251 cells transfected by pcDNA3.1-FUS

Article Snippet: The following primary antibodies were used: AKT (rabbit, Proteintech, 10176-2-AP, WB1:1500, IP:1:250, RIP:1:100); FUS (rabbit, Abcam, ab23439, WB1:2000, IP1: 200, RIP1:100); phospho-Akt (Ser473) (rabbit, Cell Signaling, #4060, WB1:1500); phospho-Akt (Thr308) (rabbit, Cell Signaling, #13038, WB1:1500); hexokinase I (rabbit, Cell Signaling, #2024, WB1:1000); hexokinase II (rabbit, Cell Signaling, #2867, WB1:1000); Flag (mouse, Sigma-Aldrich, F1804, IP 1:200); GAPDH (mouse, Sangon, D190090, WB 1:5000); H3 (rabbit, Beyotime, AH433, WB 1:500); and p53 (mouse, Active Motif, 39739, WB 1:1000, RIP 1:150).

Techniques: Activation Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Transfection, Lysis

Journal: Journal of Hematology & Oncology

Article Title: A cytoplasmic long noncoding RNA LINC00470 as a new AKT activator to mediate glioblastoma cell autophagy

doi: 10.1186/s13045-018-0619-z

Figure Lengend Snippet: LINC00470 promoted the tumorigenesis of GBM cells. a Expression levels of LINC00470 were measured by RT-qPCR in primary cultured GBM cells (LINC00470 had relatively low expression in PG-1 and PG-2; LINC00470 had relatively high expression in PG-3 and PG-4). Primary cultured GBM cells were transfected with si-LINC00470 or pcDNA3.1-LINC00470. Data are presented as the mean ± S.E.M. of three independent experiments; ** p < 0.01, *** p < 0.001. b An EDU assay was applied to assess cell proliferation of primary cultured GBM cells. Primary cultured GBM cells were transfected with pcDNA3.1-LINC00470 or si-LINC00470. c Western blotting measured the expression levels of autophagy marker LC3, beclin-1, ATG7, and ATG5 in PG-1 and PG-3 cells. The cells were transfected with pcDNA3.1-LINC00470 or si-LINC00470. d Electron microscopy detected the autophagy of U251 cells transfected with pcDNA3.1-LINC00470. e Western blotting measured the expression levels of autophagy marker LC3, beclin-1, ATG7, and ATG5 in PG-1 and PG-3 cells. The cells were transfected with si-HK1, si-FUS or si-AKT. f Survival analysis showed that Sprague Dawley rats transplanted with U251-sh-LINC00470 cells have longer overall survival. g Tumor growth for U251-sh-control and U251-sh-LINC00470 in Sprague Dawley rats.* p < 0.05, ** p < 0.01. h H&E staining showed the volume and morphology of tumors in mice transplanted with U251-sh-LINC00470 cells. The white circle represents the size of the tumor. i Western blotting measured the expression levels of the autophagy marker LC3, beclin-1, ATG7, and ATG5 in intracranial transplanted tumors. j Expression of Ki-67 and LINC00470 in intracranial transplanted tumors was detected by immunohistochemical staining or in situ hybridization, respectively

Article Snippet: The following primary antibodies were used: AKT (rabbit, Proteintech, 10176-2-AP, WB1:1500, IP:1:250, RIP:1:100); FUS (rabbit, Abcam, ab23439, WB1:2000, IP1: 200, RIP1:100); phospho-Akt (Ser473) (rabbit, Cell Signaling, #4060, WB1:1500); phospho-Akt (Thr308) (rabbit, Cell Signaling, #13038, WB1:1500); hexokinase I (rabbit, Cell Signaling, #2024, WB1:1000); hexokinase II (rabbit, Cell Signaling, #2867, WB1:1000); Flag (mouse, Sigma-Aldrich, F1804, IP 1:200); GAPDH (mouse, Sangon, D190090, WB 1:5000); H3 (rabbit, Beyotime, AH433, WB 1:500); and p53 (mouse, Active Motif, 39739, WB 1:1000, RIP 1:150).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Transfection, EdU Assay, Western Blot, Marker, Electron Microscopy, Control, Staining, Immunohistochemical staining, In Situ Hybridization