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. 2015 Nov 19;5(6):988–995. doi: 10.1016/j.stemcr.2015.10.014

Figure 2.

Figure 2

TAp73 Is Required for Metformin-Induced Self-Renewal and Proliferation of Adult Neural Precursors

(A and B) Primary neurospheres (NSs) were cultured from the SVZ of adult mice (3 months old) in the presence or absence of metformin (Met) and treated with the pan-PKC inhibitor, chelerythrine. (B) Quantification of primary neurospheres following 6 days in culture was done based on representative micrographs shown in (A). Scale bar, 200 μm. ∗∗p ≤ 0.01 (pooled data from four independent experiments).

(C) The SVZ of 3-month-old WT and CBPS436A-KI mice was dissected and cultured, and primary neurosphere formation was quantified 6 days later in the presence and absence of metformin. ∗∗p ≤ 0.01. (pooled data from three independent experiments).

(D) Metformin- or PBS-treated primary neurospheres (as in A) from both genotypes were passaged into untreated media, and the number of secondary neurospheres was quantified 4 days later. ∗∗p ≤ 0.01. (pooled data from three independent experiments).

(E) qRT-PCR for TAp73 mRNA performed on RNA extracted from primary neurospheres grown in the presence or absence of metformin. ∗∗p ≤ 0.01 (n = 4 for each group).

(F–H) Primary neurospheres were cultured from the SVZ of 3-month-old TAp73+/+ and TAp73−/− mice in either the presence or absence of 500 nM metformin. Scale bar, 200 μm. (G) Quantification of primary neurospheres from both genotypes following metformin exposure was done based on representative micrographs shown in (F). ∗∗p ≤ 0.01 (pooled data from four independent experiments). (H) Primary neurospheres cultured with or without metformin (as in A) from both genotypes were passaged into untreated media, and secondary neurospheres were counted 4 days later. ∗∗p ≤ 0.01 (pooled data from four independent experiments).

(I) Confocal micrographs of representative coronal sections through the lateral ventricles of metformin- or PBS-injected TAp73+/+ and TAp73−/− mice, stained for BrdU (green) 24 hr after BrdU injection. Sections are counterstained for Hoechst 33258 (blue). Scale bar, 100 μm.

(J and K) Quantification of the total number of BrdU-positive (BrdU+ve) cells in the SVZ (J) and SGZ (K) of metformin- or PBS-treated mice of both genotypes, as pictured in (I). ∗∗∗p ≤ 0.001; ∗∗p ≤ 0.01 (pooled data from three independent experiments).

Error bars indicate SEM.