Archives of Dermatological Research. Silencing the KCNK9 potassium channel (TASK-3) gene disturbs

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1 Archives of Dermatological Research Silencing the KCNK9 potassium channel (TASK-3) gene disturbs mitochondrial function, causes mitochondrial depolarization, and induces apoptosis of human melanoma cells 1* Dénes Nagy, 1* Mónika Gönczi, 1 Beatrix Dienes, 2 Árpád Szöır, 1 János Fodor, 1 Zsuzsanna Nagy, 1 Adrienn Tóth, 3 Tamás Fodor, 3,4 Péter Bai, 1 Géza Szücs, 5# Zoltán Rusznák, 1# László Csernoch 1 University of Debrecen, Faculty of General Medicine, Department of Physiology, Nagyerdei krt 98., Debrecen, 4012, Hungary 2 University of Debrecen, Faculty of General Medicine, Department of Biophysics and Cell Physiology, Egyetem tér 1., Debrecen, 4032, Hungary 3 University of Debrecen, Faculty of General Medicine, Department of Medical Chemistry, Egyetem tér 1., Debrecen, 4032, Hungary 4 MTA-DE Cell Biology and Signaling Research Group, Debrecen, Hungary 5 Neuroscience Research Australia, Barker Street, Randwick, Sydney, NSW, 2031, Australia *These authors contributed equally to the work. # Authors contributed equally as last authors. Send all correspondence and reprint requests to László Csernoch PhD DSc Department of Physiology, University of Debrecen, H-4012 PO Box 22, Debrecen, Hungary csl@edu.unideb.hu Phone: Fax:

2 Online Resource Figure 1: A2058 melanoma cells feature strong TASK-3-specific immunolabelling. [A] TASK-3-specific signal. [B] The same area as in [A] but presenting the distribution of MitoTracker Red to visualize mitochondria. [C] DAPI-specific signal of the same area showing the size, shape, and distribution of the cell nuclei. [D] Overlay image of [A C].

3 Online Resource Figure 2: Differentiation between the JC-1- and GFP-specific green emissions. [A1 A3] Examination of a cell culture containing GFP-expressing cells that have not been loaded with JC-1. [A1] Application of the GFP-specific settings (excitation and detection wavelengths of 488 and nm, respectively). [A2] Application of the JC-1 monomer-specific settings (excitation and detection wavelengths of 514 and nm, respectively). [A3] Application of the JC-1 J-aggregate-specific settings (excitation and detection wavelengths of 514 and nm, respectively). [B1 B3] Same as [A1 A3] but the cells were previously loaded with JC-1. The stars mark two cells which present GFPspecific emission but are not visible when either the JC-1 monomer-specific or the J- aggregate-specific setting is used.

4 Online Resource Figure 3: The effect of reduced TASK-3 expression on the MitoTracker-labelling intensity of stably transfected WM35 cells. [A1 C2] Melanoma cells were incubated with 200 nm MitoTracker Red before fixation and investigated with a laser scanning confocal microscope. Representative images taken from control [A1, A2], scrambled shrna-transfected [B1, B2], and TASK-3 knockdown cells [C1, C2]. The corresponding low- [A1 C1] and high-magnification images [A2 C2] were captured using the same confocal and camera settings. [D] Mean background-corrected fluorescence intensities in non-transfected (control), scrambled shrna-transfected, and TASK-3 knockdown melanoma cells. The numbers in the columns indicate the number of cells tested.

5 Online Resource Figure 4: Determination of the amount of total cell DNA (expressed as corrected relative fluorescence intensity) three days after seeding. The presented data reflect the results of four different experiments obtained from WM35 cells. The reduced fluorescence intensity of the knockdown WM35 cells indicates decreased DNA content of the cell culture, which is the consequence of reduced cell number with or without decreased rate of cell division.

6 Online Resource Figure 5: Morphological changes presented by TASK-3 knockdown WM35 melanoma cells. [A1 C2] Representative images taken from control [A1, A2], scrambled shrna-transfected [B1, B2], and knockdown [C1, C2] cells. [D] Proportion of cells presenting one of the three principal shapes. Representative examples of the individual shapes (labelled *, x, and +) are demonstrated in [A1], [A2], [B1], and [C2]. [E] The mean surface area of control, scrambled shrna-transfected, and TASK-3 knockdown cells. Each column represents the mean cell surface area of 50 cells.

7 Online Resource Figure 6: Reduced TASK-3 expression results in translocation of AIF from mitochondria into the cell nuclei of WM35 cells. [A1] Distribution of the AIFspecific immunolabelling in control (non-transfected) cells. [A2] The same area as in [A1] but the propidium iodide- (PI) specific signal is shown. [A3] Overlay image of [A1] and [A2]. The star indicates one of the two visible cell nuclei. Note that the AIF-specific signal is absent from the nuclei. [B1 B3] Distribution of AIF-specific immunolabelling in scrambled RNA-transfected WM35 cells. The arrangement of the images and the meaning of the star are the same as in [A1 A3]. [C1 C3] AIF-specific immunolabelling in TASK-3 knockdown WM35 melanoma cells. The star marks one of the many visible cell nuclei. Note that AIFspecific immunolabelling is present within the cell nuclei.

8 Online Resource Figure 7: In transiently transfected A2058 cells, the reduced TASK-3 expression results in translocation of AIF from the mitochondria into the cell nuclei. [A] Distribution of the AIF-specific immunolabelling in control (non-transfected) A2058 cells. [B] The same area as in [A] but the DAPI-specific signal is shown. [C] Overlay image of [A] and [B]. Note that the AIF-specific signal is absent from the cell nuclei. [D G] The arrangement of the panels is similar to that in [A C] but the images were captured from TASK-3 knockdown A2058 melanoma cells. [F] shows the distribution of the GFP-specific signal. Strong GFP expression identifies successfully transfected cells. The oblique arrow points to one of the successfully transfected cells. Note that in this cell the AIF-specific signal is present within the cell nucleus, which indicates apoptosis. In contrast, the cell marked with downward arrow has AIF-free nucleus. Because this cell does not present GFP-specific signal, it can be inferred that it is a non-transfected cell.