LIP and ROS levels in cell lines overexpressing Pfn2. (A) HeLa cells were transiently transfected with pCMV6-Kan/Neo empty vector, pCMV6-Pfn2, or Pfn2-Mut S138D vector. As control, cells were transfected with pCMV6-Fth1 vector or with the empty vector and treated with 200 µM DFO or 200 µM ferric ammonium citrate (FAC) for 16 hours. cLIP was measured by the calcein-AM method 48 hours after transfection. Data were normalized to cells transfected with the empty vector, set as 100%. Means ± standard deviation (SD) are shown. (B) Stable Hepa1-6 and HeLa clones overexpressing the WT mouse Pfn2 or Pfn2-Mut S138D were isolated, and LIP was measured and normalized to cells stably transfected with the pCMV6 empty vector, set as 100%. Means ± SD are shown. (C) ROS levels were measured using the 2′,7′-dichlorofluorescin diacetate method in HeLa cells transiently transfected with pCMV6-Kan/Neo empty vector, pCMV6-Pfn2, Pfn2-Mut S138D, or pCMV6-Fth1 vectors or in cells transfected with the empty vectors and treated with 200 µM DFO for 16 hours. ROS levels were assayed 48 hours after transfection following a pretreatment with 200 µM H₂O₂ to induce ROS generation. ROS quantifications were normalized to cells transfected with the empty vector and not treated with H₂O₂, set as 100%. Means ± SD are shown. *P ≤ .05, ***P ≤ .001. ns, not significant.