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1 Supplementary Information Figure S1. Int6 gene silencing efficiency. (A) Western Blot analysis of Int6 expression at different times after sirna transfection. Int6 expression is strongly silenced in Int6 knockdown GBM cells () compared to negative control () at 24 h, 48 h, 96 h and 7 days after sirna transfection; (B) Western Blot analysis of Int6 expression using different sirna concentrations. Int6 expression is inhibited with treatment starting at 1 nm, 5 nm and 50 nm of sirna in all glioma cell lines; (C) Western Blot analysis of HIF-1α and HIF-2α expression after Int6 inhibition. HIF-1α and HIF-2α expression is reduced in human GBM cells where Int6/eIF3e is inhibited (); (D) Western Blot analysis of Int6 and HIF-2α expression after transfection with other sirna sequences against Int6 (, Dharmacon On-Target plus Smart pool). Int6 inhibition is confirmed as well as the decreased HIF-2α expression in human GBM cells when Int6/EIF3E is silenced; (E) Western Blot analysis of Int6, HIF-1α and HIF-2α expression after transfection with from Qiagen Pool FlexiTube Gene Solution (Qiagen, Venlo, Limburg, Netherlands). Int6 inhibition is confirmed as well as the decreased HIF expression in human GBM cells when Int6/EIF3E is silenced.
2 S2 Figure S2. Int6 inhibition with different and distinct sirna sequences also alters human glioma cell proliferation. Proliferation assay with GBM cells transfected or not with (A) Int6 sirna (, Dharmacon On-Target plus Smart pool) or (B) Int6 sirna (, Qiagen Pool FlexiTube Gene Solution, Qiagen, Venlo, Limburg, Netherlands) shows a significant decreased GBM cell growth when Int6 is inhibited compared to the negative control group (* p < 0.05, ** p < 0.01, *** p < versus, n = 3).
3 S3 Figure S3. Int6 gene silencing haltes cell cycle in glioma cells. (A) Representative dot plots of cell cycle distribution in four GBM cell lines (U87,, LN18 and SF767). FL2-H represents the Propidium Iodide staining; (B) Representative dot plots of GBM cells in G0 phase. Int6 knockdown () significantly increases the number of Ki67 negative cells compared to negative control group (), n = 4. FL1-H represents Ki67 staining and FL2-H represents Propidium Iodide staining. A U87 Compter LN18 SF767 PI B U87 Ki67 LN18 SF767 PI
4 S4 Figure S4. Int6 gene silencing induces human glioma cell apoptosis. (A) Representative dot plots showing percentage of Annexin V positive cells in GBM cell lines ( and SF767). FL1-H represents Annexin V-FITC staining and FL2-H represents Propidium Iodide staining; (B) Western Blot analysis of caspase 3 and PARP expression in GBM cells following Int6 silencing. Caspase 3 and PARP expressions are reduced in Int6 knockdown group compared to negative control group (); (C) Western Blot analysis of, the pro-apoptotic protein, Bax expression in GBM cells. Bax expression increases in all glioma cell lines after transfection with different concentrations (1, 5 or 50 nm) compared to negative control group (). A SF767
5 S5 Figure S5. Int6 gene silencing does not alter global GBM cell translation. (A) Total proteins of the same number of glioma cells transfected or not with (72 h after transfection) were resolved by SDS-polyacrylamide gel and stained with blue Coomassie. Lane M, molecular weight marker; at the bottom of the gel is indicated the number of cells loaded for each cell line. De novo protein synthesis in cells was assessed, 24 h (D1), 48 h (D2) and 72 h (D3) after transfection with or control sirna () using 35 S metabolic labeling (B) and Coomassie blue staining (C), n = 3. A M U87 LN18 SF Cell # Coomassie blue staining B D1 D2 D3 C D1 D2 D3 35 S metabolic labelling Coomassie blue staining
6 S6 Figure S6. Int6 deletion affects human glioma cell migration. (A) Migration of control cells () or cells where Int6 is inhibited () was assessed using a scratch wound assay. Test was performed with LN18, SF767 and cell lines. Of note, U87 cells do not form a monolayer of cells and we were therefore unable to perform the test for these cells. Representative photographs shown in (A) were taken after the scratch (T0), and 20 h later (T20). We selected time points <24 h to avoid the effect of Int6 inhibition on proliferation (B) Percentage of wound closure was determined, 13 h (T13) and 20 h (T20) after wound, at consistent locations and was significantly decreased for cells compared to control cells at 20 h (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 3). Figure S7. Int6 gene silencing does not alter MAPK pathway in GBM cells. Western Blot analysis of MAPK pathway (MEK1, ERK and phospho-erk) in glioma cells after Int6 depletion, n = 3.
Nature Neuroscience: doi: /nn Supplementary Figure 1
Supplementary Figure 1 EGFR inhibition activates signaling pathways (a-b) EGFR inhibition activates signaling pathways (a) U251EGFR cells were treated with erlotinib (1µM) for the indicated times followed
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Supplemental Figure 1. Western blot analysis indicated that MIF was detected in the fractions of
Supplemental Figure Legends Supplemental Figure 1. Western blot analysis indicated that was detected in the fractions of plasma membrane and cytosol but not in nuclear fraction isolated from Pkd1 null
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Description of Supplementary Files. File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables
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SUPPLEMENTAL MATERIALS AND METHODS. Puromycin-synchronized metabolic labelling - Transfected HepG2 cells were depleted of
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Supplementary Figure 1: Tissue of Origin analysis on 152 cell lines. (a) Heatmap representation of the 30 Tissue scores for the 152 cell lines. The scores summarize the global expression of the tissue
a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p.
a. b. c. d. e. f. g. h. i. j. k. l. 2.5 2 1.5 1.5 IL-1β 12 8 6 4 2 IL-1β 9 8 7 6 4 3 3 2.9 IL-1β m. n. o. p. 1.8 1.6 1.4 1.2 1.8.6.4.2 6h LPS 2 15 1 5 6h LPS 2 6h LPS 6 4 3 6h LPS Supplementary Figure
Int6/eIF3e Is Essential for Proliferation and Survival of Human Glioblastoma Cells
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Supplementary Figure 1
Supplementary Figure 1 6 HE-50 HE-116 E-1 HE-108 Supplementary Figure 1. Targeted drug response curves of endometrial cancer cells. Endometrial cancer cell lines were incubated with serial dilutions of
Supplementary Materials and Methods
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File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables. File Name: Peer Review File Description:
File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables File Name: Peer Review File Description: Primer Name Sequence (5'-3') AT ( C) RT-PCR USP21 F 5'-TTCCCATGGCTCCTTCCACATGAT-3'
Supplementary Information File
Supplementary Information File Supplementary Table 1. List of synthesized sirna sequences for target genes sirna Species Sequence Ctrl sirna mouse sense 5 -UUCUCCGAACGUGUCACGUTT-3 Antisense 5 -ACGUGACACGUUCGGAGAATT-3
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Supplementary Figures
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