Fig 2: Functional assessment of ETNPPL expression in glioblastoma cell lines. (A) WB for ETNPPL in proteins extracted from Gli4, Gli7 and LGG85 cells infected with control (luciferase) or ETNPPL inducible-lentiviruses and cultured with and without doxycycline. The ETNPPL protein is only detected in ETNPPL-infected cells in the presence of doxycycline. β-actin detection is used as loading control. The uncropped image of the WB is presented on Supplemental Fig. 8B. (B) Immunofluorescence for ETNPPL in Gli7 cells infected with an ETNPPL lentivirus and cultured with and without doxycycline. ETNPPL (red) is strongly detected in the cell nuclei. Infected cells are detected by expression of the GFP (green) gene included in the lentiviral construct. (C) Representative experiment of the effect of ETNPPL overexpression on glioblastoma cell growth. Histograms show the relative number of Gli7 and LGG85 cells cultured for 8 days with and without doxycycline. Test = One-way ANOVA tests. n = 3 independent experiments. (D) Representative experiment of EdU incorporation in Gli7 cells. Histograms represent the % of EdU+ cells obtained after 4 hours of incorporation in the presence or absence of doxycycline. test = Mann-Whitney test (*p = 0.024), n = 3 independent experiments.

Fig 3: Detection of ETNPPL protein in human cortex. (A) Immunofluorescence for ETNPPL (green) in human cortex sections. The two middle photographs are high magnification of red dotted-line square on the left-hand image. ETNPPL is detected in the cytoplasm (yellow arrow) or the nucleus (red arrow). The right-hand image shows the absence of detection of ETNPPL when the antibody was pre-incubated with the ETNPPL peptide used to generate the antibody. Remaining green dots are likely to be autofluorescent lipofuscin which is often present in human brain samples. Scale bars = 20 μm. (B) Immunofluorescences for ETNPPL (green) and indicated proteins (red) in human cortex. ETNPPL is detected in the nucleus of cells expressing ALDH1L1, CHI3L1, GFAP and VIM (vimentin), identifying these cells as astrocytes. Arrows point to double positive cells. Scale bars = 10 μm.

Fig 4: ETNPPL protein reduction in foci. (A) Representative photographs of ETNPPL immunohistochemistry (brown stainings with DAB) in 3 grade II oligodendrogliomas and 4 grade II astrocytomas. Photographs were taken in the foci and the rest of the tumour (DLGG). Nuclei are stained with hematoxylin. Scale bars = 50 μm. (B) Quantification of ETNPPL+ cells in the 3 oligodendrogliomas and 4 astrocytomas. The histograms represent the % of ETNPPL+ cells detected among all cells present per 10,000 μm² for each tumour (n = 5 fields). Tests = Mann-Whitney tests. (C) Immunohistochemistry for ETNPPL (brown) in one glioblastoma. The protein is not detected. Scale bars = 150 μm. Nuclei are stained with hematoxylin.

Fig 5: Expression of ETNPPL by tumoral cells. (A) Representative photograph of double immunofluorescence for IDH1R132H/ETNPPL in one grade II oligodendroglioma. White arrows point to IDH1R132H positive tumour cells co-expressing ETNPPL in the cytoplasm (green). Red arrow points a nuclear ETNPPL expression in a non tumoral cell (IDH1R132H negative) indicative of staining specificity. Scale bars 10 μm. (B) Representative photograph of double immunofluorescence for ATRX/ETNPPL in one grade II astrocytoma. Most of ETNPPL+ cells do not express ATRX and are thus tumoral. The red arrow points to an ETNPPL+ ATRX+ cell which is unlikely to be tumoral whereas the white arrow shows an ETNPPL+ ATRX- tumoral cell. Insets are high magnification of pointed cells. Scale bars 10 μm. (C) Quantification of tumoral ETNPPL+ cells. Histograms show the average number of indicated cells by counted field (x40) in one grade II oligodendrogliomas and two grade II astrocytomas. In oligodendroglioma, ETNPPL+ cells are also IDH1 R132H+ whereas in the two astrocytomas they are ATRX-. Tests = Mann-Whitney tests are non-significant (n.s.) p = 0.74; 0.55; 0.53 for the 3 tumours. (D) Detection of ETNPPL (green) in 2 diffuse low-grade gliomas cultures. The presence of tumoral cells is indicated by staining for the mutated IDH1 R132H protein (red). Insets are high magnification of cells pointed with arrows. Note that in those two patients, the ETNPPL protein was mainly detected in the cytoplasm. Scale bars = 20 μm.