1. RhoB Promotes Cancer Initiation by Protecting Keratinocytes from UVB-Induced Apoptosis but Limits Tumor Aggressiveness 
Nicolas Meyer, Alexis Peyret-Lacombe, Bruno Canguilhem, Claire Médale-Giamarchi, Kenza Mamouni, Agnese Cristini, Sylvie Monferran, Laurence Lamant, Thomas Filleron, Anne Pradines, Olivier Sordet, Gilles Favre 
Journal of Investigative Dermatology 
Volume 134, Issue 1, Pages (January 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
The rhob-/- mice develop fewer skin tumors after chronic exposure to UVB. The rhob+/+, rhob+/- and rhob-/- mice were chronically exposed to UVB (0.1 J cm−2). (a) Mice were separated into two groups according to the percentage of actinic keratoses (AKs) on the skin of their back at week 17: group A (>50%) and group B (<50%). (b) Percentage of mice of the two groups according to their genotype. (c) Number of tumors (keratotic, ulcerated) per mouse at the indicated times (mean+SD). AK lesions were excluded. (d) Number of keratotic tumors at the indicated times (mean+SD). (e) Number of ulcerated tumors per mouse after 22 weeks (mean+SD). Examples of AK and tumors (keratotic, ulcerated) are presented in Supplementary Figure S1 online. P-values are calculated as described in the Statistical Analysis section of the Materials and Methods.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
RhoB (Ras homolog gene family, member B) is induced in human healthy skin explants after UVB exposure. (a) RhoB immunostaining on human healthy skin explant. The expression of RhoB is weak and mainly localized in basal layer keratinocytes. (b) RhoB immunostaining on human skin explants from two healthy donors (scale=20 μm), unirradiated or 8 hours after 0.4 J cm−2 UVB irradiation. The expression of RhoB is markedly increased and detectable in the entire epidermis.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Keratinocytes deficient for RhoB (Ras homolog gene family, member B) are hypersensitive to UVB-induced apoptosis. (a) Mice of the indicated genotype were exposed to a single UVB dose (0.2 J cm−2). TUNEL staining. Epidermis (ED) and dermis (D) are separated by the dotted white line (scale=5 μm). Graph represents the percentage of TUNEL-positive cells in the epidermis (mean+SD; two fields per slide; from 71 to 244 cells per field). (b) Percentage of cleaved caspase-3 in UVB-induced mice tumors (mean+SD; two fields per slide; from 199 to 584 cells per field). (c, d) Normal human keratinocytes (NHKs) were exposed to UVB (0.4 J cm−2) and analyzed 24 hours later. (c) Percentage of cleaved caspase-3-positive NHKs (mean+SD). (d) PARP (poly (ADP-ribose) polymerase) cleavage. (e) NHKs plated on coverslip were exposed to UVB (0.4 J cm−2) and then stained for 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL 16 hours after exposure. Ctrl, control; si, small interfering RNA.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
RhoB (Ras homolog gene family, member B) protects keratinocytes from UVB-induced apoptosis through Bax/B-cell lymphoma 2 (Bcl-2) ratio modulation. (a) Normal human keratinocytes (NHKs) were treated with small interfering RNA (siRNA) RhoB 1 (si 1) or 2 (si 2) or control siRNA (Ctrl) for 24 hours. Western blotting analyses of Bax and Bcl-2 at 8 and 24 hours after UVB exposure (0.4 J cm−2). (b) Bax/Bcl-2 band density ratio (mean+SEM). (c) NHKs were treated with siRNA RhoB 1 (si 1) or 2 (si 2) or Ctrl for 24 hours. Western blotting analyses of phospho-AKT (ser472/473/474), AKT, and Bcl-2 at 4 and 8 hours after UVB exposure (0.4 J cm−2). The presented blots are representative of two independent experiments on NHK primary cultures from two different donors.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
RhoB (Ras homolog gene family, member B) loss is associated with the preferential induction of undifferentiated skin tumors. Histopathological analysis and CK1, CK8, and Ki67 immunostaining were performed on formalin-fixed, paraffin-embedded (FFPE) samples of all tumors resulting from chronic exposure of the mice (rhob -/-: n=23; rhob+/+: n= 17). (a) Example representative of pathological examination, and CK1 and CK8 immunostaining of tumors (scale=10 μm). (b) Percentage of low-to-undifferentiated squamous cell carcinomas (SCCs) among all mouse tumor samples. (c) Ki67 immunostaining (scale=10 μm). The graph represents the percentage of Ki67-positive cells in the tumors (mean+SD; from 189 to 607 cells per field). (d) Quantification of RhoB mRNA in cells from tumors resulting from chronic exposure of wild-type (WT) mice (normalized as 2e−(CqRhoB-CqActin); mean+SD).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
RhoB (Ras homolog gene family, member B) loss is associated with increased levels of DNA double-strand breaks. (a) RhoB immunostaining on human squamous cell carcinomas (SCCs). Graph shows the proportion of tumors expressing RhoB depending on the level of differentiation. Diff., differentiated; Undiff., undifferentiated. (b) Phosphorylated histone H2AX (γH2AX) immunostaining on human SCCs (scale=5 μm). Graph shows the percentage of γH2AX-positive cells depending on the level of differentiation (mean+SD). (c) The 53BP1 immunostaining on human SCCs (scale=5 μm). Graph shows the percentage of positive cells depending on the level of differentiation (mean+SD). Percentages of (d) γH2AX-positive cells and (e) 53BP1-positive cells in mouse tumors depending on rhob genotype (mean+SD). (f) Proportion of γH2AX-positive keratinocytes in the epidermal basal layer (mean+SD). (g) γH2AX and 53BP1 dual staining on rhob-/- mouse healthy skin, confocal imaging.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions