Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 6

1.
Figure 2

Figure 2. Stable overexpression of GFP-tagged K8 phospo-mutants in K8 knockdown AW13516 cells.. From: Loss of Keratin 8 Phosphorylation Leads to Increased Tumor Progression and Correlates with Clinico-Pathological Parameters of OSCC Patients.

(A) Western blot analysis of stable overexpressed K8 wild type (K8WT-1 and -2), K8-Ser73Ala phospho-mutant (K8S73A-1 and -2), K8-Ser431Ala phospho-mutant (K8S431A-1 and -2) and pEGFP (pEGFP-1 and -2) clones derived from K8 knockdown cells with antibodies to K8 and K18. β-actin was used as loading control. (B) RT-PCR analysis of K8 in stable K8 wild type and phospho-mutant clones. GAPDH was used as internal control. (C) Representative confocal images of filaments formed by GFP-tagged K8 in stable K8WT-1, KS73A-1 and K8S431A-2 clones. pEGFP-N3 empty vector transfected (pEGFP-1) clone was taken as control. Scale bar: 50 µm.

Hunain Alam, et al. PLoS One. 2011;6(11):e27767.
2.
Figure 3

Figure 3. Analysis of K8 phosphorylation at Ser73 and Ser431 and their filament formation with K18 in stable K8 phospho-mutant clones.. From: Loss of Keratin 8 Phosphorylation Leads to Increased Tumor Progression and Correlates with Clinico-Pathological Parameters of OSCC Patients.

(A and B) Western blot analysis of stable overexpressed K8 wild type (K8WT-1 and -2), K8 Ser73Ala phospho-mutant (K8S73A-1 and -2) and K8 Ser431Ala phospho-mutant (K8S431A-1,-2 and -3) clones with antibodies against K8 or phospho-specific K8 Ser73 or Ser431. K8-knockdown (shRNAK8.2-C1) and vector control (pTU6-Cont) clone were taken as controls. β-actin was used to ensure equal loading. (C) Representative confocal micrographs of filaments formed by GFP-tagged K8 with endogenous K18 in K8 wild type (K8WT-1), K8Ser73Ala (K8S73A-1) and K8Ser431Ala (K8S431A-2) clones. Stably overexpressed empty vector clone (pEGFP-1) was taken as control. Scale bar: 50 µm.

Hunain Alam, et al. PLoS One. 2011;6(11):e27767.
3.
Figure 1

Figure 1. Generation of RNAi resistant K8 phospho-mutants constructs.. From: Loss of Keratin 8 Phosphorylation Leads to Increased Tumor Progression and Correlates with Clinico-Pathological Parameters of OSCC Patients.

(A) Strategy for generation of shRNA resistant K8 wild type and K8 phospho-mutants, Ser73Ala and Ser431Ala constructs. (B) Strategy for generation of K8 wild type and K8 phospho-mutants, Ser73Ala and Ser431Ala stable clones in K8-knockdown AW13516 cells. (C and D) Mutated RNAi resistant GFP-tagged K8 phospho-mutants, K8-S73A-GFP-R and K8-S431A-GFP-R were validated by cotransfecting them with shRNAK8.2 or vector pTU6-PURO in HEK293 cells. shRNA-sensitive GFP-tagged phospho-mutants (K8-S73A-GFP and K8-S431A-GFP) were also transfected separately in HEK293 cells and used as controls. Representative images of GFP expression 60 hrs of post-transfection (combination of transfection is indicated in figure; Magnification 10X). (E and F) Western blot analysis of proteins extracted from cells transfected with shRNA-resistant (K8-S73A-GFP-R and K8-S431A-GFP-R) and their respective shRNA-sensitive constructs (K8-S73-GFP and K8-S431-GFP) using K8 antibody (order of samples is indicated in figure). β-actin was used as loading control.

Hunain Alam, et al. PLoS One. 2011;6(11):e27767.
4.
Figure 6

Figure 6. IHC analysis of K8 phosphorylation in human OSCC.. From: Loss of Keratin 8 Phosphorylation Leads to Increased Tumor Progression and Correlates with Clinico-Pathological Parameters of OSCC Patients.

(A) Levels of K8 phosphorylation at Ser73 and Ser431 in K8 positive paraffin embedded section of human OSCC samples were determined by immunohistochemical (IHC) analysis. Representative images of IHC staining with site specific phospho-antibodies against K8Ser73, K8Ser431 and total K8. (Magnification: 200X). (B) Histograms showing correlations of K8 dephosphorylation at Ser73 and Ser431 with clinico-pathological parameters such as tumor size, lymph-node metastasis and tumor stage of human OSCC patients. (C and D) Kaplan–Meier analysis of overall survival in OSCC patients and loss of K8 phosphorylation at Ser73 or Ser 431 residue (for K8 Ser73 phosphorylation * P = 0.047).

Hunain Alam, et al. PLoS One. 2011;6(11):e27767.
5.
Figure 5

Figure 5. Loss of K8 phosphorylation resulted in increased tumorigenicity of AW13516 cells.. From: Loss of Keratin 8 Phosphorylation Leads to Increased Tumor Progression and Correlates with Clinico-Pathological Parameters of OSCC Patients.

(A) 103 cells from K8 wild type (K8WT-1 and -2), K8 Ser73Ala phospho-mutant (K8S73A-1 and -2) and K8 Ser431Ala phospho-mutant (K8S431A-1 and -2) clones were injected subcutaneously into 6 different NOD-SCID mice and tumor volume measured as described. Representative images of SCID mice bearing tumors of K8WT-1, K8S73A-1 and K8S431A-2 clone 60 days after the injection. Scale bar: 10 mm (B) Bars, mean value and SD of tumor volume of K8 wild type (K8WT-1 and -2), K8-Ser73Ala (K8S73A-1 and -2) and K8-Ser431Ala (K8S431A-1 and -2) mutant after 60 days is plotted. Statistical analysis was done by Student's t test. *P<0.05; **P<0.01. (C) Representative images of immunohistochemical staining (with antibodies against GFP, K8-phospho-Ser73 and K8-phospho-Ser431 as indicated) of tumor tissues obtained from NOD-SCID mice injected with K8WT-1, K8S73A-1 or K8S431A-2 clones. TBS was used as a control (Magnification: 200X). Note that K8S73A-1 and K8S431A-2 clone showed loss of K8 phosphorylation at Ser73 and Ser431 respectively.

Hunain Alam, et al. PLoS One. 2011;6(11):e27767.
6.
Figure 4

Figure 4. Effect of decreased K8 Ser 73 or Ser431 phosphorylation on migratory ability of OSCC cells.. From: Loss of Keratin 8 Phosphorylation Leads to Increased Tumor Progression and Correlates with Clinico-Pathological Parameters of OSCC Patients.

(A) Representative fluorescence and phase contrast micrographs of K8 wild type (K8WT-1) and K8 phospho-mutants (K8S73A-1 or K8S431A-2) cells that displayed GFP-tagged K8 analyzed by fluorescence microscopy. Note that the cells expressing high levels of K8-Ser73Ala or K8-Ser431Ala mutant appeared on the edges of the colony. Scale bar: 50 µm. (B) Scratch wound healing assays were performed on K8 wild type (K8WT-1), K8-Ser73Ala (K8S73A-1) and K8-Ser431Ala (K8S431A-2) clones. Phase contrast images (10X) of wound closure at 0 hr (panels a–c), 2.5 hr (panels d–f), 5 hr (panels g–i), 7.5 hr (panels j–l), and 10 hr (panels m–o) of the clones are shown in the figure. Scale bar: 100 µm. Migration rate was calculated by AxioVision software. The data shown is the average from three independent experiments with the mean and standard deviation. *P<0.05, ** P<0.01 (by student t-test).

Hunain Alam, et al. PLoS One. 2011;6(11):e27767.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk