Fig 2: COX4-1 glioma cells form neurosphere-like tumor spheroids expressing neural stem cell markers(A) Representative phase contrast photomicrographs (10× magnification) of parental U251, U251-shRNA-COX4-2, U251-TgCOX4-1, and U251-TgCOX4-2 cells after 10 days of culture in serum-free Neurobasal medium supplemented with EGF and FGF. (B) Spheroids of U251-TgCOX4-1 cells were immunostained with antibodies against COX4-1, BMI1, or CD133 or with control antibodies. (C) In vitro limiting dilution assays and quantification of COX4-1 and BMI1 expressing cells. Results represent the average from two independent experiments. (D) Spheroid multipotency was assessed by immunofluorescence for neuronal (neurofilament, CNPase, and βIII-tubulin) and glial (GFAP) markers.

Fig 3: COX4-1 and BMI1 co-expression is required to promote cell proliferation(A) Representative western blot depicting BMI1 expression in nuclear extracts of U251-TgCOX4-1 cell following 24-h PTC-209 treatment (0–10 μM). (B) Cell proliferation in control and PTC-209-treated (5 μM) U251-TgCOX4-1 cells. (C) Representative western blot depicting BMI1 expression in U251-TgCOX4-1 cells expressing shRNA control or one of four different vectors expressing shRNA against BMI1. (D) Quantification of the relative expression levels of BMI1 detected in (C). (E) Cell proliferation in clones expressing shRNA against BMI1. (F) Representative western blot depicting BMI1 expression levels (inset) and the cell proliferation rates of control and pCMV6-BMI1-transfected U251 cells. Graphs represent the average from triplicate determinations from at least three independent experiments.

Fig 4: Mitochondrial ROS regulates BMI1 expression(A) Representative histograms from flow cytometric analysis of total cellular ROS (left, DCFDA fluorescence) and mitochondrial ROS (right, MitoSOX fluorescence) in parental and U251-TgCOX4-1 cells. Bar graphs provide quantitative analysis of fluorescence intensity. (B) Quantitative graphs showing the relative levels of catalase activity, superoxide dismutase activity, NAD+/NADH ratio, and GSH/GSSG ratio in U251-TgCOX4-1 cells. (C) Representative histograms from flow cytometric analysis of total cellular or mitochondrial ROS production in U251-TgCOX4-2 cells treated with NAC (300 μM) or PTC-209 (5 μM) (left) and in U251-TgCOX4-1 cells treated with NMP (10 μM) or PTC-209 (right). (D) Representative western blots depicting BMI1 expression in the nuclear extracts of parental cells or U251-TgCOX4-1 cells after treatment with NAC or PTC-209 for 24 h (top) and quantitative analysis of expression levels (bottom). Bars represent the average from triplicate determinations from at least three independent experiments.

Fig 5: COX4-1 drives proliferative capacity in human glioma cells(A) COX4-1 and COX4-2 constructs ( pCMV6-COX4-1-FLAG and pCMV6-COX4-2-FLAG) were transfected into U251-COX4-2 depleted cells to create U251-TgCOX4-1 and U251-TgCOX4-2 stable cell lines. Expression of COX4 isoforms and BMI1 was detected in each cell line by western blot analysis. Citrate synthase (CS) expression is shown as mitochondrial loading control and actin expression is shown as nuclear loading control. (B) Proliferation rates of each cell line. (C) Representative pictures of clonogenic assays with each cell line, showing anchorage-independent cell growth. (D) Representative images of tumors from athymic nude mice inoculated with the cell lines. Tumors were excised 4 weeks after inoculation. (E) Analysis of tumor volumes in mice over the course of the experiment. (F) Comparison of tumor weights upon excision. Graphs represent the average from triplicate determinations from at least three independent experiments.