Fig 2: Manipulation of ADK isoforms affects morphology, migration, and invasion of cultured MDA-MB-231 cells. (A) Cell morphology of MDA-MB-231 cells with manipulation of either ADK-L knockdown (MBA-ADK-LD), ADK-S knockdown (MBA-ADK-SD), or without ADK changes (MBA-ADK-WT), in their early, middle, and late passages. The MBA-ADK-LD cells look bigger and flatter, while a necrotic morphology was observed on the MBA-AKD-SD cells. (B) Both knockdowns of ADK-L (MDA-ADK-LD) and ADK-S (MDA-ADK-SD) reduced migration of cultured MDA-MB-231 cells without ADK modification (MDA-ADK-WT). (C) Knockdown of ADK-S (MDA-ADK-SD) reduced the invasion efficiency of MDA-MB-231 (MDA-ADK-WT) cells (D) Anchorage-independent colony formation assay and (E) representative images of cell colonies of MDA-MB-231 cells with modification of ADK isoforms. ** p<0.01; *** p<0.001; **** p<0.0001.

Fig 3: Expression profiles of ADK isoforms in cancer tumor versus NAT from 46 patients. (A) Representative Western blot of 6 TNBC patients’ cancer tissue (Ca) versus paired normal adjacent tissue of tumor (NAT). (B) Quantitative analysis of ADK-L and ADK-S expressions of pooled subtypes of breast cancer (a total of 46 patients): Luminal A and B positive, Her2+, and TNBC subtypes. (C) Quantitative analysis of ADK-L and ADK-S expression in 19 luminal A and luminal B positive subtype patients (left panel), 14 TNBC subtype patient (middle panel); and 13 Her2+ subtype patients (right panel). * p<0.05; *** p<0.001; red line indicates normalized 1-fold expression level of ADK in NATs.

Fig 4: ADK expression levels. (a, left) Western blots showing the expression levels of ADK in whole brain homogenates of saline (sal) and KA injected Panx1f/f, NFH-Cre:Panx1f/f, and GFAP-Cre:Panx1f/f mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. (a, right) Relative mean ± SEM values of ADK expression levels obtained from densitometric analysis of western blots of brain homogenates displayed in the left panel. The values of p were obtained from one-way ANOVA followed by Tukey’s multiple comparison test. (b, left) Representative images of brain sections immunostained using anti-ADK antibodies obtained from saline- and KA-injected Panx1f/f and GFAP-Cre:Panx1f/f mice. Note that high levels of immunoreactivity following KA treatment in GFAP-Cre:Panx1f/f compared with Panx1f/f mice. (b, right) Relative mean ± SEM values of ADK fluorescence obtained from images of brain sections of Panx1f/f, GFAP-Cre:Panx1f/f, and NFH-Cre:Panx1f/f mice 40 to 60 min after KA-induced bilateral forelimb clonus (square) compared with saline injected mice (circles). The values of p were obtained from two-way ANOVA followed by Sidak’s multiple comparison test. N = number of mice; GFAP = glia fibrilary acid protein; NFH =neuro filament H ; KA = kainic acid; ADK = adenosine kinase.

Fig 5: ADK manipulations in breast cancer cells and its effect on growth and viability. (A) MCF 10A (non-cancerous breast cell line) and (B) MDA-MB-231 (breast cancer cell line) cell proliferation comparison after ADK-L and ADK-S CRISPR transfection. 1 million cells seeded 24h prior to transfection in 6-well plates; cell count performed after Ki-67 staining (n=20). (C) Growth curve of cultured MDA-MB-231 cell lines as quantified by Trypan Blue cell counting. Both ADK-L and ADK-S knockdown mutant cells have longer doubling times than unmodified MDA-ADK-WT cells (D) Cell viability over time was quantified by MTT assay. As measured formazan absorbance is directly proportional to the number of viable cells, it can be seen that viability is decreased in both modified cell lines.