Correction: Renal Cells Express Different Forms of Vimentin: The Independent Expression Alteration of these Forms is Important in Cell Resistance to Osmotic Stress and Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0068301.]

Renal Cells Express Different Forms of Vimentin: The Independent Expression Alteration of these Forms is Important in Cell Resistance to Osmotic Stress and Apoptosis

<div><p>Osmotic stress has been shown to regulate cytoskeletal protein expression. It is generally known that vimentin is rapidly degraded during apoptosis by multiple caspases, resulting in diverse vimentin fragments. Despite the existence of the known apoptotic vimentin fragments, we demonstrated in our study the existence of different forms of vimentin VIM I, II, III, and IV with different molecular weights in various renal cell lines. Using a proteomics approach followed by western blot analyses and immunofluorescence staining, we proved the apoptosis-independent existence and differential regulation of different vimentin forms under varying conditions of osmolarity in renal cells. Similar impacts of osmotic stress were also observed on the expression of other cytoskeleton intermediate filament proteins; e.g., cytokeratin. Interestingly, 2D western blot analysis revealed that the forms of vimentin are regulated independently of each other under glucose and NaCl osmotic stress. Renal cells, adapted to high NaCl osmotic stress, express a high level of VIM IV (the form with the highest molecular weight), besides the three other forms, and exhibit higher resistance to apoptotic induction with TNF-α or staurosporin compared to the control. In contrast, renal cells that are adapted to high glucose concentration and express only the lower-molecular-weight forms VIM I and II, were more susceptible to apoptosis. Our data proved the existence of different vimentin forms, which play an important role in cell resistance to osmotic stress and are involved in cell protection against apoptosis.</p></div

Differential expression of VIM forms under time-dependent osmotic stress conditions.

<p>Western blot analysis of cytoskeletal proteins during osmotic stress adaptation. A: TALH-STD cells were stressed with 30 mM glucose medium and tested for protein expression changes after 0, 24, 48, 72, and 96 h with antibodies against the indicated protein. B: TALH-STD cells were stressed with 600 mosmol/kg NaCl medium and analyzed after 0, 24, 48, 72, and 96 h. C: Adapted TALH-Glu cells were switched back to hypoosmolar medium with 300 mosmol/kg medium and tested for protein expression changes after 0, 24, 48, and 72 h. D: Adapted TALH-NaCl cells were switched back to hypoosmolar (300 mosmol/kg) medium and tested for protein expression changes after 0, 24, 48, and 72 h. E: 2D western blot analysis of VIM expression in TALH-STD cells in the course of hyperosmolar glucose stress. TALH-STD cells were stressed with 30 mM glucose medium and tested for VIM expression after 0, 24, 48, 72, and 96 h. VIM IV subtype (arrowhead) increases after 24 h during hyperosmolar glucose stress and then decreases to almost absent in TALH-Glu cells that are cultured more than 96 h in glucose media. Immunofluorescence staining of VIM in TALH cells. F: cells switched from Glucose to hypoosmolar medium for 24, 48, and 72 h, the scale bars represent 10 µm. G: cells switched form 600 mosmol/kg NaCl to hypoosmolar medium for 24, 48, and 72 h, the scale bars represent 5 µm. Immunofluorescence staining of cells was then carried out using anti-VIM antibody. Image analysis was carried out as described in material and method part.</p

Differential expression of VIM forms under osmotic stress conditions in TK173 renal fibroblast cells.

<p>A: 2D western blot analysis of VIM expression in TK173 cells during hyperosmolar NaCl stress. After 72 h of NaCl stress, 4 VIM forms (numbered I–IV) appeared. B: Immunofluorescence staining of TK173 cells during NaCl stress. After 48 h, VIM builds a strong filamentous network around the nucleus, shown with a green arrowhead. The scale bars represent 3 µm.</p

Expression of VIM and cell viability analysis of TALH cells during apoptosis induction.

<p>Western blot analysis of vimetin and lamin A/C expression in A: TALH-Glu and B: TALH-NaCl cells after 0, 2, 4, 6, and 8 h of treatment with 100 ng/ml TNF-α together with 10 µg/ml cycloheximide. C: Cell viability, determined with MTT assay, showed an increased survival in TALH-NaCl cells compared to TALH-STD and TALH-Glu cells in a time-dependent manner from 0 to 8 hr with TNF-α treatment. D: TALH-Glu and E: TALH-NaCl cells in the course of apoptosis induction with 1 µM staurosporine. F: Cell viability, determined with MTT assay, showed an increased resistance in TALH-NaCl cells compared to TALH-STD and TALH-Glu cells in a time-dependent manner from 0 to 8 hr with TNF-α treatment. VIM bands of higher molecular weight appeared after 2 h of treatment with TNF-α and staurosporine in TALH-Glu cells. In addition, lamin A/C was cleaved in a 28-kDa fragment (arrowhead) by caspase activation after 4 h. However, TNF-α and staurosporine treatments showed neither expression change in VIM forms nor cleavage products of lamin A/C in TALH-NaCl cells.</p

VIM knock-down using siRNA.

<p>A: Western blot analysis of VIM in non-transfected TALH-STD and TALH-STD cells transfected with the VIM siRNAs 1, 2, 3 or all three combined. B: siRNA transfected TALH-STD-cells and TALH-STD were subjected to either to NaCl or glucose stress. Cell viability and proliferation, determined with MTT assay, showed a slight increase in cell proliferation upon siRNA treatment but the transfected cells showed higher sensitivity to osmotic stress compared to control. C: The monitoring of apoptosis in siRNA TALH-STD-cells subjected to osmotic stress was carried out using Western blot for caspase 8 (left panel) and 3 (right panel). Each bar represents means ± S.D. of results from 3 independent experiments. Significant differences: (*) p<0.05, (**) p<0.01, (***) p<0.001.</p

Differential expression of VIM forms under osmotic stress conditions in HK2renal epithelial cells.

<p>A: 2D western blot analysis of VIM expression in HK2-STD and HK2-NaCl cells in the pI range 4–7. Lower forms of VIM showed a decrease in expression in HK2-cells adapted to high NaCl. B: Immunofluorescence staining of VIM in HK2-NaCl cells showed an extended network compared to HK2-STD cells the scale bars represent 8 µm.</p

Western blot analysis of VIM after inhibition of caspases or proteases in TALH cells.

<p>A: TALH-cells adapted to high glucose (lane 1: control) were treated for 2 h with the pan-caspase inhibitor z-VAD-fmk (lane 2) and then 4 h with 1 µM staurosporine (lane 3) or 100 ng/ml TNF-α plus 10 µg/ml CHX (lane 4). B: TALH-cells adapted to high NaCl (lane 1: control) were treated for 2 h with the pan-caspase inhibitor z-VAD-fmk (lane 2) and then 4 h with 1 µM staurosporine (lane 3) or 100 ng/ml TNF-α plus 10 µg/ml CHX (lane 4). TALH cells (lane 1: control) were treated for 4 h with TLCK (lane 2) or TPCK (lane 3). C: TLCK but not TPCK influences the expression of VIM forms in TALH-Glu cells. D: TALH- NaCl showed no change in the expression of VIM forms. β-actin antibody was kept as control. The molecular mass in kDa is indicated at the left of each panel.</p

Differential expression regulation of cytoskeletal proteins under different osmotic stress conditions.

<p>A: Enlargments of gel regions in the range of pH 5–8 that were cropped from Flamingo fluorescence dye stained 2D-gels. The panels show differential expression of VIM forms in control cells (TALH-STD) and cells cultured in hyperosmolarity medium of NaCl (TALH-NaCl) and glucose (TALH-Glu). Quantitative analyses were carried out by comparing the VIM expression changes between TALH-STD and –NaCl and between TALH-STD and –Glu. The expression quantification of the spots is presented as a grouped bar chart with error bars. Each bar represents the intensity means ± S.D. of vimentin protein spots from 3 independent experiments. Significant differences: (*) p<0.05, (**) p<0.01, (***) p<0.001. B: Ci Western blot analysis of cytoskeletal proteins VIM, CK, and CFL in TALH-STD, TALH-NaCl, TALH-Glu, and TALH-Urea cells. VIM showed 4 different bands, called forms I (46 kDa), II (48 kDa), III (50 kDa), and IV (54 kDa), from lower to higher molecular weight, respectively. VIM forms showed differential regulation under different stress conditions, where β-actin, used as control, was equally expressed. Cii the WB bands from the different VIM forms (I–IV) were quantified separately. CK showed four different bands in Western blot, all the four bands were up-regulated in TALH-NaCl. In TALH-Glu cells only the form with lowest molecular weight showed slightly up-regulation, when compared to TALH-STD. CFL expression level was significantly down-regulated in TALH-NaCl –Glu. The expression quantification is presented as a grouped bar chart with error bars on the left. Each bar represents the intensity means ± S.D. of blots from 3 independent experiments. Significant differences: (*) p<0.05, (**) p<0.01, (***) p<0.001. C: Immunofluorescence staining of TALH-cells using mouse anti-VIM (a, b, c, d), anti-CK (e, f, g, h), and anti- CFL (i, j, k, l) antibodies in TALH-STD, TALH-NaCl, TALH-Glucose, and TALH-Urea cells, respectively. VIM builds a strong filamentous network in TALH-NaCl (b) cells compared to strong perinuclear restriction in TALH-Glu (c) cells. The number of cells with perinuclear localisation of the stained protein (VIM, CK or CFL) was counted and presented in per cent of the total number of cells in the wells. The stressed cells were compared with the TALH-STD. Each bar represents the number of cells with perinuclear localization in % (± S.D of 200 counted cells/well from 3 independent wells. Significant differences: (*) p<0.05, (**) p<0.01, (***) p<0.001. Arrows indicate the cell with perinuclear localisation of IFs. In case of (J) the arrows indicate the perinuclear localisation of CFL. All the scale bars represent 20 µm; D: TALH-STD, -NaCl- and -Glu cells, respectively, were seeded on 96-well plates and allowed to attach for 24 h. The cells were processed as described in material and methods. Slices were contrasted with uranyl acetat and lead citrate. Analysis of the section was done with a LEO906 E electron microscope. Right and left panel represents same cells with different magnification n: nucleus, m: mitochondria G: golgi apparatus, Δ: plastic foil (where the cells attach), arrow: indicate the border of the cells *: indicate the IFs localization. All the scale bars on the left panel represent 2 µm and on the right panel represent 5 µm.</p

List of differentially expressed proteins identified in TALH-Glu cell line compared to TALH-STD cells (pH range 5–8).

<p>List of differentially expressed proteins identified in TALH-Glu cell line compared to TALH-STD cells (pH range 5–8).</p