Fig 1: Expression of genes related to cell death in rods (a) and cones (b). Bar plots showing transcriptomic changes in genes coding for phosphodiesterase 6 (PDE6; i.e., Pde6a, Pde6b, Pde6c, Pde6g, Pde6h), cyclic-nucleotide-gated channels (CNG channels; i.e., Cnga1, Cngb1, Cnga3, Cngb3), protein kinase G (PKG; Prkg1, Prkg2), poly(ADP-ribose) polymerase (PARP; Parp1, Parp2), histone deacetylase (HDAC; Hdac1, Hdac2, Hdac3), Na+/Ca2+ exchanger 1 (NCX1; Slc8a1), cAMP response element-binding protein 1 (Creb1) and cAMP response element modulator (Crem), and apoptosis inducing factor 1 (Aifm1). The x-axis indicates postnatal day (P), and y-axis depicts average log2 fold change; positive values indicating higher expression in rd1 rods/cones. Bars were color-coded (red for rods; blue for cones) for p-values < 0.05.
Fig 2: Proposed mechanism of the deleterious action of ISM1 on podocytes. (Left) Caspase-dependent pathway. At low concentration, extracellular ISM1might bind avß5 and/or GRP78 at the cell surface, triggering activation of procaspase-8 and the downstream effector caspase-3, which would activate a caspase-dependent DNAse (CAD) to produce DNA fragmentation (left). At higher concentrations, ISM1 is internalized by endosomal trafficking and would induce the activation of a caspase-independent proapoptotic pathway. Thus, ISM1 potentially interacts with GRP78 on the cell surface and would trigger its endocytic transport into the mitochondria. Subsequently, ISM1 triggers the permeabilization of the outer mitochondrial membrane and the specific release of AIF into the cytosol. Ultimately, AIF translocates to the nucleus where it contributes to DNA fragmentation and chromatin condensation.
Fig 3: ISM1 induces apoptosis in podocytes. (a) Estimation of ISM1-induced apoptosis by TUNEL assay after un strong stimulus ISM1 (35 µg/mL) for 72 h. Cells were observed under fluorescence microscopy. (b) Apopotic cells were quantified. ISM1 induced an emergence of TUNEL positive cells corresponding to apoptotic cells (** p < 0.01 vs control group; n = 3). (c,d) Evidence of caspases activation. Representative immunoblots with quantitative data of densitometry are shown in (c) for caspase 8 and in (d) for caspase 3. ISM1induced the degradation of pro-caspases 8 and 3 (** p< 0.01; * p < 0.05 vs control group; n = 4 blots) this was inhibited by QVD-OPh. (e) ISM1 decreases the mitochondrial membrane potential of podocytes (??m). Podocytes were treated with ISM1 (35 µg/mL) and quantification of ??m was assessed by the JC-1 probe. ISM1 decreased significantly the ??m of podocytes from 48 h to 72 h (*** p < 0.01; ** p < 0.01 vs control group). (f) AIF was released from mitochondria to the cytosol and the nucleus after ISM1 treatment (arrows). In contrast, cytochrome c remained confined in mitochondria after ISM1 treatment. Bars = 50 µm for (a) and 20 µm for (f).
Fig 4: BA increases AIF nuclear localization and expression of AIFsh in PC cells. A. Western blot analysis showing increase in nuclear AIF (57 kd) in LNCaP (48 h) and DU145 (72 h) cells treated with MB compared to M, B, and control (C) cells. B. Western blot analysis of total protein lysates showing increase in AIFsh (35 kd) in MB, DB, and B treated LNCaP (48 h), DU145, and PC3 (72 h) compared to M, D, or C. Coomassie blue stain of total protein is loading control. C. Immunofluorescence showing increase in nuclear localization of AIF in DU145 cells treated with DB compared to C cells (72 h). In control DU145 cells, AIF (green) is cytoplasmic and the merge with nuclear PI stain (red) shows little nuclear localization. In DB treated DU145 cells, the merge of AIF and PI shows some nuclear localization (arrows; yellow). (×200). D. RT-qPCR analysis showing increase in AIFsh mRNA in LNCaP cells treated 48 h with MB, DB, or B alone compared to M or D alone. AIFsh mRNA levels were relative to C = 1. n = 5, three independent experiments; *, P < 0.05. E. Knockdown of AIF reduces cell death in LNCaP cells treated with MB and DB. Trypan blue exclusion assay showing that LNCaP/shAIF-2 (AIF) cells undergo less cell death compared to control LNCaP/shGFP (C) after treatment for 24 h with MB or DB. n = 6, three independent experiments; *, P < 0.02. Western blot analysis showing knockdown of AIF and AIFsh proteins in LNCaP/shAIF-2 results in lower cleaved PARP after MB or DB treatment compared to control LNCaP/shGFP cells. Coomassie blue stain of total protein is loading control.
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