Figure 5.

Elevated DNA Damage Response in FL-Derived HSCs against Culture-Induced Stress

(A–D) Bone marrow and E14.5 FL-derived KLS cells were cultured in serum-free medium in the presence of SCF and Tpo for 5 days. Expression for DDR genes from the harvested cells (after culture [AC]) was compared with the freshly isolated (before culture [BC]) LSK cells by performing qRT-PCR. Culture-induced change in the expression of genes that belong to different DDR pathways was plotted. (A) mismatch repair, Pcna, Rpa3, Rfc4, Rfc5; (B) nucleotide excision repair, Cetn2, Ddb1; (C) base excision repair, Neil1, Neil3, Xrcc1, Tdg; (D) homologous recombination, Topbp1, Palb2, Mre11a, Brca1, Blm. n = 4–6, t test: ^∗p < 0.05.

(E and F) Neutral comet assay was performed using freshly isolated and cultured KLS cells from BM and FL tissues. Olive tail moment (E) and proportion of cells with comet tail (F) was compared for different samples. n = 4, N > 223, one-way ANOVA followed by Tukey's multiple comparisons test: ^∗p < 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.005.

(G) Immunostaining for γ-H2AX was performed on freshly isolated and cultured LSK cells from BM and E14.5 FL tissues. Scale bar, 5 μm.

(H) Proportion of cells with γ-H2AX for was compared between different samples. n = 4, N > 173, one-way ANOVA followed by Tukey's multiple comparisons test: ^∗p < 0.05, ^∗∗p ≤ 0.01.

See also Figure S5.