Extended Data Figure 3. Cre-mediated depletion of PTEN-targeting microRNAs in astrocytes.
a, IHC analyses of AMPK, Bim, and E2F1 expression (mean ± s.d, t-test) in brain metastasis tumors with/without pre-knocking out (KO) miR-17-92 cluster in brain microenvironment. M: brain metastases; B: brain tissue. b, Schematic of experimental design. GFAP-Cre adenovirus (Ad-GFAP-Cre) was injected intracranially to the right hemisphere of the Mirc1 mouse, and the control adenovirus (Ad-βGLuc) was injected intracranially to contralateral side of the brain. Then B16BL6 cells were injected intracranially to both sides. c, IHC analysis of Cre expression in the brain astrocytes. d, IHC analysis of PTEN expression in the tumor cells. e, Quantifications of PTEN expression in tumor cells (mean ± s.d., t-test). f, Quantification of intracranial tumor outgrowth by volume (mean ± s.e.m., t-test). g, qRT-PCR analyses of miR-19a and PTEN mRNA in tumor cell HCC1954 after 48 hour co-culture with primary astrocytes from Mirc1tm1.1Tyj/J mice pre-infected (48 hours) by adenovirus (Ad-βGLuc or Ad.GFP.Cre) (mean ± s.e.m., t-test). h, WB of PTEN protein in the indicated tumor cells co-cultured as in (g). i, Knockout of miR-17-92 allele in cultured primary astrocytes. miR-17~92 cluster is flanked by loxP site in Mirc1tm1.1Tyj/J mouse. Primary astrocytes were isolated from Mirc1 mouse brain then infected by adenovirus encoding for βGLuc or GFP.Cre protein. Concentrated adenovirus particles of indicated volume (same MOI ~108 units/mL) encoding βGLuc or GFP.Cre proteins were added to 106 astrocytes. Left, representative photo showing the infection efficiency. Right: bar diagram showing the relative miR-19a expression (one of the five miRNA genes in the miR-17-92 cluster) three days after adenovirus infection (mean ± s.e.m., t-test).