1. c-Rel Downregulation Affects Cell Cycle Progression of Human Keratinocytes 
Verena N. Lorenz, Michael P. Schön, Cornelia S. Seitz 
Journal of Investigative Dermatology 
Volume 134, Issue 2, Pages (February 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Expression of c-Rel in squamous cell carcinoma (SCC). (a) Staining of c-Rel in paraffin-embedded tissue of normal skin, (b) invasive SCC, and (c and d) Bowen’s disease. Note positive c-Rel staining of (d, arrows) mitotic figures. (a–c) Bar=250 μm; (d) bar=125 μm. (e) Quantitative analysis of c-Rel staining in normal epidermis (NL), actinic keratosis (AK), Bowen’s disease (BD), and invasive SCC; error bars are ±SD; ***P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Downregulation of c-Rel leads to growth arrest and increase of apoptosis in HaCaT keratinocytes. (a) Western blotting of c-Rel in control (ctrl) and c-Rel small interfering RNA (siRNA)–transfected cells after 72 hours. (b) Phase-contrast photographs 72 hours after transfection. Bar=100 μm. (c) Growth curve of control siRNA (dashed line) and c-Rel siRNA cells (continuous line) from 24 hours until 96 hours after transfection. At least six microscopic fields (original magnification, × 160) were evaluated. (d) MTT-based proliferation assay 72 hours after siRNA transfection. (e) BrdU proliferation assay 72 hours after transfection showing significant inhibition of proliferation in c-Rel-downregulated HaCaT cells. (f) Apoptosis assay 72 hours after transfection. For all experiments: one representative of three independent experiments is shown, each experiment was performed in triplicates; error bars are ±SD; ***P<0.001, **P<0.01, *P<0.05. Ctrl, control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
c-Rel downregulation leads to an increased number of HaCaT cells in the G2/M phase and induces phosphorylated-histone H3Ser10 (p-H3Ser10) expression. (a) Cell cycle phase distribution of cells determined by FACS analysis. (b) Cell cycle phase distribution of vital control (gray) and c-Rel (black) small interfering RNA (siRNA) cells of four independent experiments. (c) Western blotting of G1/S and G2/M regulatory proteins for control (ctrl) and c-Rel siRNA-transfected HaCaT cells. (d) Digitally enhanced p-H3Ser10 immunofluorescence staining of cells counterstained with 4′,6-diamidino-2-phenylindole. Bar=50 μm. (e) Evaluation of p-H3Ser10–positive cell portion of cells 72 hours after transfection. At least 10 microscopic fields (original magnification, × 100) were evaluated. Two independent experiments yielded similar results. For all evaluations, error bars are ±SD; **P<0.01, *P<0.05. Ctrl, control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
c-Rel silencing causes increased mitotic cells as well as aberrant mitotic spindle formation. (a) Photographs of control (ctrl) and c-Rel knockdown cells stained for β-tubulin 72 hours after transfection. Arrows indicate aberrant mitotic spindles classified as monopolar, and arrowheads show bipolar abnormal mitotic spindles. Bar=20 μm. (b) Evaluation of abnormal spindle formation of control (ctrl) and c-Rel small interfering RNA (siRNA)–transfected cells 72 hours after transfection. Classification of spindle morphology based on immunofluorescence findings. One of two similar evaluations is shown here. (c) Evaluation of aberrant mitotic spindle portion of ctrl and c-Rel siRNA cells subdivided into mono-, bi-, or multipolar spindle morphology. Classification based on immunofluorescence findings. One representative of three independent experiments is shown, and at least 19 microscopic fields were evaluated; error bars are ±SD; ***P<0.001, **P<0.01. (d) Western blotting of aurora A and phospho-aurora AThr288 (p-aurora AThr288) for ctrl and c-Rel siRNA-transfected HaCaT cells. (e) Double immunostaining of control and c-Rel knockdown cells for β-tubulin and p-aurora AThr288. Bottom row shows merged picture with β-tubulin, p-aurora AThr288, and 4′,6-diamidino-2-phenylindole (blue). Bar=20 μm. Ctrl, control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions