Fig 2: Pluri-IQ Application Pipeline and Its Performance Evaluation in Immunofluorescence Images(A) The main graphical user interface (GUI) of Pluri-IQ.(B) GUI used to select different folders containing the images to perform manual validation, autoscoring or data comparison.(C) Pluri-IQ pipeline: two different images with different degrees of pluripotency were used to create the machine-learning training set (upper panel). After each channel segmentation and colony identification, a fluorescence training set was created and manually validated. After the classifier automatic update, two new images were scored automatically by Pluri-IQ and classification accuracy was evaluated (bottom panel). Scale bar, 500 µm (in raw images). Color code on the raw images: green, actin; red, OCT4. Color code on the images prediction: green, pluripotent colonies (Plur); blue, mixed colonies (Mix); red, differentiated colonies (Dif).(D) Percentage of pluripotent, mixed, and differentiated colonies in the two different experimental conditions. Data derived from the automatic data comparison in Pluri-IQ. Results derived from two replicates. LIF, pluripotency medium; N2B27, neuronal differentiation medium.See also Figure S2.

Fig 3: IGF2 is regulated by stemness factor. (A) IGF2 expression was detected by an mRNA array between tumorsphere (TS) and adherent (2D) cells. In the three liver cancer cell lines (Hep3B, HuH7, and HepG2), IGF2 mRNA (A) and protein (B) levels were measured with qRT-PCR and an immunoblot assay. Blots were cropped to show specific bands. (C) IGF2 expression was examined during sphere culture (3, 5, 7 days). (D-E) IGF2 mRNA (D) and protein (E) expression was detected after transient transfection of stemness factor (NANOG, OCT4, and SOX2). For qRT-PCR, relative expression was calculated using 18 s expression as an internal control. Immunoblots used ß-actin as a loading control. Blotted proteins were visualized by enhanced chemiluminescence (GE Healthcare, UK) with the automatic X ray film Imaging System (Konica Minolta, Japan). Blots were cropped to show specific bands. The average (column) ± SD (bar) is indicated (***p < 0.001).

Fig 4: PRMT7 and its catalytic activity are indispensable for the stemness of mESCs, and Oct4, Nanog, Klf4 and c-Myc protein levels are more reduced than their mRNA levels in PRMT7-depleted mESCs. (A) Microscopic and AP-staining images of shLuc-treated and PRMT7-depleted V6.5 mESCs. Five shPRMT7s (shPRMT7-4, -5, -6, -7 and -8) were used to deplete PRMT7. BF, bright field; AP, alkaline phosphatase; red scale bar, 100 μm. (B) The percentages of shLuc- or shPRMT7-treated V6.5 mESCs in G1, S and G2/M phase of the cell cycle. shLuc-treated cells were used as a control. (C) Rescue experiments of PRMT7-depleted mESCs with PRMT7 or its catalytic mutant (m.PRMT7). shPRMT7-7 or shPRMT7-8 plasmids were co-electroporated with expression plasmids encoding both GFP and either PRMT7 or its catalytic mutant. The GFP-expressing vector pCDH1-EF1-IRES-GFP (vector/GFP) was used to clone PRMT7 cDNA. Microscopic, green-fluorescent and AP staining images of indicated group of cells are shown (left panel). Western blotting was used to analyze the expression levels of PRMT7 or its mutant in eight different experimental conditions (right panel). (D) Analysis of Oct4, Nanog, Sox2, c-Myc and Klf4 mRNA levels in shLuc-treated and PRMT7-depleted V6.5 mESCs using quantitative RT-PCR. (E) The effect of ectopic expression of PRMT7 or its catalytic mutant (m.PRMT7) on Oct4, Nanog, Sox2, c-Myc, Klf4 and Prmt7 mRNA levels in PRMT7-depleted mESCs. Relative Oct4, Nanog, Sox2, c-Myc, Klf4 and Prmt7 mRNA levels were examined in eight different experimental conditions (see panel C for lane description). Red scale bar, 100 μm. (F and G) Western blot analysis of Oct4, Nanog, Sox2, c-Myc, Klf4 and PRMT7 levels in shLuc-treated and PRMT7-depleted V6.5 mESCs. Oct4, Nanog, Sox2, c-Myc, Klf4 and PRMT7 protein levels in shLuc-treated and PRMT7-depleted V6.5 mESCs were quantified (G). Data are presented as the mean ± SD of three independent experiments. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) indicate statistically significant changes.

Fig 5: 4-AAQB inhibits the nuclear localization of ß-catenin, Sox2, and Oct4 in GBM cells. U87MG and DBTRG-05MG cells treated with or without 5–10 µM 4-AAQB were immunostained with Sox2 (green), Oct4 (red), ß-catenin (red), and F-actin (green) antibodies, then image visualization and analysis carried out by fluorescence microscopy. Treatment with 5 or 10 µM 4-AAQB markedly decreased nuclear Sox2 and Oct4 protein expression in (A) U87MG or (B) DBTRG-05MG cells. (C) Immunohistochemistry show reduced nuclear expression of ß-catenin and F-actin after treatment with 10 µM 4-AAQB. Original magnification ×200. DAPI (blue) served as nuclear marker.