Fig 7. Expression of HMGA2 affects human subtelomeric domains upon induced replication stress.

(A) Southern blot analysis of H1299 cells (parental and HMGA2 KO cell line) after PFGE separation using telomere-specific probes. SN38 treatment at three different doses as indicated was done for 48 h, with DMSO used as control. (B) Western blot showing exogenous expression of HMGA2 (top panel) after transfection with 1.5 μg HMGA2 expression vector in H1299 HMGA2 KO cells (mock vector served as control). Southern blot analysis of HMGA2 KO transfected cells after PFGE separation using telomere-specific probes (bottom panel). 2 μM SN38 treatment was done for 48 h, with DMSO used as control (n = 3 independent transfection experiments followed by SN38 treatment). (C) Representative Southern blot analysis of HeLa cells (parental and three recombinant HMGA2-expressing cell lines) after PFGE separation using telomere-specific probe. 2 μM SN38 treatment was for 48 h, with DMSO as control (n = 3 independent experiments). (D) Western blot showing HMGA2 expression for indicated cell lines with β-actin used as internal control (top panel). Southern blot analysis of human embryonic stem (hES) cells after PFGE separation using telomere-specific probes (bottom panel). 0.1 μM SN38 treatment was for 24 h, with DMSO as control (n = 3 independent experiments). (E) Representative Southern blot analysis of HeLa cells (parental and three recombinant HMGA2-expressing cell lines) after PFGE separation using telomere-specific probe. 100 mM HU treatment was done for 24 h (n = 3 independent experiments). (F) PFGE analysis of DSB formation in HeLa cells (DNA stained with EtBr) in response to 24 h incubation with indicated doses of TMPyP4 (top panel) and the corresponding Southern blot analysis using telomere-specific probes (bottom panel). 100 mM HU treatment for 24 h was used as control for comparing telomeric DNA fragments (>1 Mb and 30–100 kb fractions).