Journal: Scientific Reports
Article Title: Disruption of intracellular iron homeostasis through mitochondrial dysfunction associated with suppression of ATP 13A2 expression
doi: 10.1038/s41598-026-35368-x
Figure Lengend Snippet: Protective effect of ATP13A2 KD in α-Syn-SH cells by inhibiting iron influx. Apo Transferrin or Glibenclamide (GBD) were administered for 24 h after ATP13A2 KD in α-Syn-SH cells. ( A ) Atomic absorption spectrometry for evaluation of total intracellular iron level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.579, NC apo-Tf = 1.645, siATP = 2.671, siATP apo-Tf = 1.984). ( B ) Staining of RhoNox-4 for evaluation of intracellular Fe 2+ level in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( C ) Quantification of (B) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 156, 89, 93, 94, 116, Cell number of NC apo-Tf for each biological replicate: 146, 101, 117, 91, 76, Cell number of siATP for each biological replicate: 90, 134, 94, 108, 126, Cell number of siATP apo-Tf for each biological replicate: 135, 103, 68, 120, 112, NC = 1.0, NC apo-Tf = 1.069, siATP = 1.510, siATP apo-Tf = 1.121). ( D ) MitoSOX dye for detection of mitochondrial ROS in α-Syn-SH cells treated with apo Transferrin for 24 h (0.10 mg/mL) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( E ) Quantification of (D) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 203, 180, 99, 163, Cell number of NC apo-Tf for each biological replicate: 236, 223, 208, 199, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP for each biological replicate: 187, 188, 175, 145, Cell number of siATP apo-Tf for each biological replicate: 162, 181, 173, 166, NC = 1.0, NC apo-Tf = 1.013, siATP = 2.729, siATP apo-Tf = 1.552). ( F ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with apo Transferrin for 24 h (0.01, 0.10, 0.50, or 1.00 mg/mL) after ATP13A2 KD ( n = 4, biological replicates, NC = 100, siATP = 78.34, siATP apo-Tf 0.01 mg/mL = 91.84, siATP apo-Tf 0.10 mg/mL = 102.3, siATP apo-Tf 0.50 mg/mL = 105.0, siATP apo-Tf 1.0 mg/mL = 101.8). ( G ) Atomic absorption spectrometry in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD. Quantification of iron content per 1.0 × 10 5 cells ( n = 3, biological replicates, NC = 1.276, NC GBD = 1.130, siATP = 1.882, siATP GBD = 1.268). ( H ) Staining of RhoNox-4 in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: Rho-Nox4, Blue: Hoechst) (Scale bar, 10 μm). ( I ) Quantification of (H) ( n = 5, biological replicates, Cell number of NC for each biological replicate: 73, 52, 60, 77, 54, Cell number of NC GBD for each biological replicate: 62, 86, 99, 96, 94, Cell number of siATP for each biological replicate: 123, 65, 80, 115, 97, Cell number of siATP GBD for each biological replicate: 82, 64, 81, 127, 95, NC = 1.0, NC GBD = 1.073, siATP = 1.849, siATP GBD = 1.126). ( J ) MitoSOX dye in α-Syn-SH cells treated with Glibenclamide for 24 h (30.0 µM) after ATP13A2 KD (Red: MitoSOX, Blue: Hoechst) (Scale bar, 20 μm). ( K ) Quantification of (J) ( n = 4, biological replicates, Cell number of NC for each biological replicate: 121, 119, 153,128, Cell number of NC GBD for each biological replicate: 115, 158, 125, 139, Cell number of siATP for each biological replicate: 106, 109, 83, 99, Cell number of siATP GBD for each biological replicate: 119, 122, 128, 97, NC = 1.0, NC GBD = 0.904, siATP = 2.325, siATP GBD = 1.839). ( L ) Cell viability was measured by CCK-8 assay in α-Syn-SH cells treated with Glibenclamide for 24 h (3.0, 10.0, or 30.0 µM) after ATP13A2 KD ( n = 6, biological replicates, NC = 100, NC GBD 10 µM = 118.1, siATP = 73.66, siATP GBD 3 µM = 87.66, siATP GBD 10 µM = 94.94, siATP GBD 30 µM = 95.94). Each value represents the mean ± SEM. An ANOVA, followed by the Bonferroni/Dunn post-hoc test was used to test the significance of differences (n.s. not significant. * p < 0.05, ** p < 0.01, *** p < 0.001). siATP: siRNA targeting ATP13A2, NC: siRNA of negative control.
Article Snippet: The transferred membrane was incubated in 5% skim milk (Nakarai Tesque) or Blocking One (Nakarai Tesque) at room temperature for 60 min. After blocking, the membrane was incubated with the following primary antibodies: the mouse monoclonal antibody transferrin receptor (1:500, Invitrogen), and β-actin (1:2000, Santa Cruz Biotechnology); rabbit polyclonal antibodies: ATP13A2 C-terminal region (1:1000, Sigma-Aldrich), LC3 (1:1000, MBL), SQSTM1/p62 (1:1000, Cell Signaling), IRP2 (1:1000, Novus Biologicals), ferritin and DMT1 (1:1000, Abcam) α-Synuclein (1:2000, Abcam) dissolved in 5% skim milk or Reagent A of Immuno-enhancer (FUJIFILM) at 4 °C overnight.
Techniques: Staining, CCK-8 Assay, Negative Control
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