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First published online 25 January 2005
doi: 10.1242/jcs.01647

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Characterization of human epiplakin: RNAi-mediated epiplakin depletion leads to the disruption of keratin and vimentin IF networks

Shyh-Ing Jang^*,,, Alexandr Kalinin^*, Kaoruko Takahashi, Lyuben N. Marekov and Peter M. Steinert^¶

Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, MD 20892-8023, USA

View larger version (85K): [in a new window]   Fig. 1. Immunoblotting analyses and the distribution of epiplakin in cultured cells. (A) Immunoblot of NHEK-L (lane 2) and Cos-7 (lane 3) extracts probed with an antibody to epiplakin. ß-tubulin is shown as a loading control. (B) Equal amounts of the soluble (S) and insoluble (P) fractions were resolved in a 3% SDS-NuPAGE gel and transferred to PVDF membrane, probed with the anti-epiplakin (1:2000) and the HRP-conjugated anti-rabbit antibodies (1:20,000). Nebulin, with a molecular mass of 850 kDa, is marked. (C) Immunofluorescent staining of HeLa (a), A431 (b), HaCaT (c) and NHEK-L (d) cells. Cells were fixed in cold methanol and stained with anti-epiplakin antibody. The thin filament structure is indicated by arrows in d. Bars, 20 µm.

View larger version (126K): [in a new window]   Fig. 2. Co-localization of epiplakin with keratin and vimentin IF networks. (A) Triple-label immunostaining in HeLa cells with antibodies to epiplakin (epip), vimentin (vim) and keratin (kerat). The co-localization of epiplakin and vimentin appears yellow and is indicated by arrowheads. Co-localization of the three proteins appears white (arrows). Bars, 20 µm. (B) Immunostaining of epiplakin (epip) and keratin 14 (K14) in HaCaT cells. The intense epiplakin staining is indicated by arrowheads (a). Boxed area in c is shown at higher magnification in d-f. The branch structure of the network is indicated by arrows. Bars, 10 µm. (C) Immunostaining of keratinocytes maintained in low calcium (a-c) and high calcium medium (one day; d-f). Cells were fixed and stained with antibody of epiplakin (epip) and keratin 5 (K5). Arrows indicate co-localization. Bars, 20 µm.

View larger version (69K): [in a new window]   Fig. 3. Up-regulation of epiplakin expression in keratinocytes. (A) Immunofluorescent staining of NHEK cells maintained in low calcium (a) and high calcium for 1 day (b) or high calcium for 3 days (c), fixed and stained with anti-epiplakin (red) antibody and TO-PRO 3 for nuclei (blue). Bars, 20 µm. (B,C) Immunoblotting of soluble (B) and insoluble (C) fractions from NHEK-L (lane 2) or NHEK-H of 1 (lane 3), 3 (lane 4), 5 (lane 5), 7 (lane 6) and 9 days (lane 7) were probed with epiplakin antibody. ß-tubulin is shown as a loading control. (D) Real-time RT-PCR analysis of epiplakin and involucrin transcripts in NHEK cells. The threshold values (Ct) of epiplakin and involucrin were normalized by the subtraction of the Ct value of ß-actin in each corresponding samples (insert: epiplakin, black line; involucrin, blue line). Three separate experiments were performed with triplicate samples. The data are presented as a fold change (mean ± s.d.) of the relative level of epiplakin (black and white bars) and involucrin (hatched bar) from NHEK-H or HeLa over that of NHEK-L sample.

View larger version (39K): [in a new window]   Fig. 4. Overlay binding assay of epiplakin fragments with vimentin, keratins and assembled IFs. (A) Schematic illustration of human epiplakin 9 [according to the nomenclature of Fujiwara et al. (Fujiwara et al., 2001)]. (B) Dot-blot binding assay of recombinant purified monomeric or polymerized IF proteins with expressed epiplakin fragments (right: schematic representation of the GST-fused epiplakin constructs). The indicated recombinant proteins were spotted in duplicate onto three membranes as described in Materials and Methods. The expressed GST-EpiL12B13 protein (lane 1) served as a positive control for GST antibody. Protein loading was monitored by Amido Black staining. The experiments were repeated in at least three times and one representative set of data is shown.

View larger version (81K): [in a new window]   Fig. 5. Knock down of epiplakin by RNAi. (A) Immunostaining of HeLa cells transiently transfected with siRNA-SC (a) and siRNA-KD (b). Cells were fixed and stained with the anti-epiplakin (epip) antibody. Nuclei were stained with TO-PRO 3 (blue). Bars, 30 µm. (B) Western blotting analysis of epiplakin expression in HeLa cells transiently transfected with siRNA-SC or siRNA-KD (2, 5, 10, 25, and 50 nM). Cellular extracts were fractionated on a 3% SDS-NuPAGE gel and probed with anti-epiplakin antibody. ß-actin is shown as a loading control. The level of epiplakin was measured by densitometry and normalized to ß-actin in each corresponding sample. The data (mean ± s.d.) are averages from three separate experiments with duplicate samples and are presented as a fold change from the sample of siRNA-KD over that of siRNA-SC. (C) Immunostaining analysis of HeLa cells transiently transfected with siRNA-KD. (a-d) Cells were subjected to triple-label staining with the antibodies specific for epiplakin, vimentin and keratin. (e,f) In a parallel experiment, cells were double-stained with antibodies for keratin (kerat) and ß-actin (actin). The non-transfected cells are indicated by arrows (a-f). Bars, 20 µm.

View larger version (103K): [in a new window]   Fig. 6. The knock down of epiplakin in epidermal cells. (A) Immunofluorescent staining of epiplakin in NHEK transiently transfected with the indicated RNA duplex and maintained in either low calcium (NHEK-Low; a,b) or high calcium (NHEK-Hi; c,d) medium for 2 days, fixed and stained with anti-epiplakin antibody (red). Nuclei were stained with TO-PRO 3 (blue). Bars, 20 µm. (B) Immunofluorescent staining in HaCaT cells transiently transfected with 50 nM of siRNA-SC (a) or siRNA-KD (b,c) were stained with anti-epiplakin (epip; a,b) and anti-keratin (kerat; c). b and c are from the same field. Bars, 20 µm.

View larger version (71K): [in a new window]   Fig. 7. The effects of stable knock down of epiplakin on cytoskeletal structures. (A) Immunoblotting analysis of HeLa stable cell lines. Equal amounts of cellular extracts from two stable cell lines of pSilent/Epi-SC (lane 1, 2) or pSilent/Epi-KD (lane 3, 4) were fractionated on a SDS-NuPAGE gel and probed with anti-epiplakin antibody. The molecular mass of nebulin (850 kDa) is marked. (B) Immunofluorescent staining of the stable HeLa cell lines. The stable epiplakin knockdown cells were stained with the antibodies to epiplakin (epip), keratin (kerat), and also with either anti-ß-actin (actin) antibody (a-c), or anti-ß-tubulin (tub) antibody (d-f). The control (g-i) was stained with antibodies of epiplakin (epip), keratin (kerat) and vimentin (vim). a-c, d-f and g-i are from the same field. Bars, 20 µm.

View larger version (65K): [in a new window]   Fig. 8. The effects of epiplakin linker and B domain on the IF network in the absence of epiplakin. (A) Immunofluorescence analysis of HeLa cells transiently transfected with siRNA-KD together with pEGFP-B13 (a-c), pEGFP-L12 (d-f) or pEGFP-L12B13 (g-i). Cells were fixed and double-stained with anti-vimentin (vim; b,e,h) and anti-keratin (kerat; c,f,i) antibodies. a, d and g show the green fluorescence signal of EGFP. Panels a-c, d-f and g-i are from the same field. Bars, 20 µm. (B) Immunofluorescence analysis of stable epiplakin knockdown HeLa cell lines. Cells were transiently transfected with pEGFP-L12B13 and were double-stained with anti-epiplakin (epip; b,e), anti-keratin (kerat; c) or anti-vimentin (vim; f) antibodies. a and d show the green fluorescence signal of EGFP. a-c and d-f are from the same field. Bars, 20 µm.

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