Oligos for Real-Time RT-PCR experiments.

<p>Oligos for Real-Time RT-PCR experiments.</p

Wnt5a is expressed in the embryonic epidermis but not required for hair follicle induction.

<p>(A) Wnt5a highly expressed in the epidermis, and its expression in the dermis is relatively weak, no significant expression is detected in developing placodes. (B and C) <i>Shh</i> is normally expressed in placodes in Wnt5a null embryos at E14.5. (D and E) The down-growth of embryonic hair follicles in <i>Wnt5a</i>-deficient skin is unaffected, as indicated by red arrows. Abbreviations: epi, epidermis; p, placode.</p

Hair follicle placode formation is impaired without Wls.

<p>(A and D) <i>Shh</i> positive placodes are decreased in <i>Wls</i>-deficient skin compared with the control. (B and E) <i>Bmp4</i> expressing dermal condensates are reduced in <i>K14-cre;Wls^c/c</i> mutants. (C and F) <i>Lef1</i> positive placodes decrease significantly in <i>Wls</i>-deficient skin. (G) Statistical analysis of the <i>Shh</i>, <i>Bmp4</i> and <i>Lef1</i> positive spots in embryo skin. **, P<0.01; *, P<0.05. cKO, <i>K14-cre;Wls^c/c</i>.</p

Assessment of autophagy in high over-expressing line.

<p>(A), Representative images of Immunostaining of LC3 on paraffin section of hearts from high over-expressing line and WT control mice. (B), Quantification of LC3 positive dots (For each group, 4 mice were studied). (C), Representative western blot of autophagy-related proteins LC3, Beclin 1, LAMP-1 and cathepsin D in heart extracts obtained from 3-month-old high over-expressing line and WT control mice. (D), Densitometric analysis of LC3 immunoblots. (E), Representative western blot of time course analysis of LC3 processing in heart extracts obtained from high over-expressing line and WT control mice. (F), Representative western blot of acetylated p53 and DRAM.</p

Echocardiographic analysis of cardiac function of high over-expressing line.

<p>(A), Representative M-mode echocardiography images in 3-month-old mice. (B)–(C), LV end-diastolic diameter (LVID;d) and LV end-systolic diameter (LVID;s) were significantly increased in high over-expressing line compared to WT control mice. (D), Left ventricular posterior wall (LVPW) was significantly decreased in high over-expressing line compared to WT control mice. (E)–(F), % fractional shortening and ejection fraction were significantly diminished in high over-expressing line compared to WT control mice. *<i>P</i><0.05, WT vs. TG (n = 5 for TG, n = 5 for WT, 3 months old).</p

<i>K14-cre;Wls^c/c</i> mice display patched hair loss.

<p>(A–B) Hairless patches are present in <i>K14-cre;Wls^c/c</i> and <i>K14-cre;Wls^c/c</i> mice. (C–D) Hair follicles disappear in hairless patches in <i>K14-cre;Wls^c/c</i> mutant skin (the black arrow), whereas hair follicles are maintained in hairy patches but usually decrease in number and are progressively lost with aging. (E–G) K14-cre activity is not evenly distributed in the skin. Black arrows indicate placodes, and red arrows indicate inter-placode basal layers. (H–K) Wls protein is significantly reduced in the surface ectoderm at E13.5 and hairless patches at P17 in <i>K14-cre;Wls^c/c</i> skin, indicating effective deletion of Wls by K14-cre. Abbreviations: epi, epidermis; der, dermis.</p

The effect of hPDCD5 over-expression on the survival of mice.

<p>(A), Autopsy of transgenic founder 41 showing dramatically enlarged heart. (B), Kaplan-Meier Survival Curves of high over-expressing line (red line) and WT littermate controls (black line). (n = 50 for WT, n = 65 for TG).</p

Up-regulation of PDCD5 in Ang II-induced cardiac hypertrophy.

<p>(A), HW∶BW ratio showing significant increase in cardiac mass in mice treated with Ang II for 2 weeks compared to sham control mice. (B) Expression of ANF was determined by quantitative Real-Time RT-PCR analysis in cDNA samples derived from hearts of mice treated with Ang II and sham control. (C), Expression of βMHC was determined by quantitative Real-Time RT-PCR analysis in cDNA samples derived from hearts of mice treated with Ang II and sham control. (D), Representative western blot of PDCD5 and internal control actin proteins in heart extracts from 8–week-old male mice with sham or Ang II treatment. (E), Quantitative analysis revealed that PDCD5 levels were up-regulated in hearts from mice treated with Ang II (n = 4) as compared to sham control mice (n = 4). *<i>P<0.05</i>, sham vs. Ang II treatment.</p

<i>Wls</i> is dynamically expressed during hair follicle induction and hair cycling.

<p>(A) Wls is expressed both in the surface ectoderm and underlying mesenchyme of the E13.5 embryo. (B) Wls expression is sustained in the epidermis and appears in nascent placodes at E14.5. (C) Wls is detected in the growing placodes and epidermis at E15.5. (D) <i>Wls</i> is expressed in the bulge region, second hair germ, and dermal papilla in early anagen. (E) Expression of <i>Wls</i> in full anagen. High-level <i>Wls</i> expression is observed in matrix keratinocytes and pre-hair shaft, the expression in dermal papilla becomes weak. The dermal sheath has no significant <i>Wls</i> expression. (F) Expression of <i>Wls</i> in early catagen. Wls is strongly expressed in ORS, IRS, and hair shaft, the expression in dermal papilla is increased. (G) Expression of Wls begins to decrease in mid-catagen, especially in epithelial strands. (H) Wls expression in late catagen mainly exists in lower hair follicles and the expression in dermal papilla is significant. (I) In telogen, Wls expression is mainly maintained in the bulge region, expression in dermal papilla is decreased. Bar = 30 µm. Abbreviations: b, bulge; d, dermal papilla; ds, dermal sheath; epi, epidermis; es, epithelial strand; h, hair shaft; i, inner root sheath; m, matrix; o, outer root sheath; ph, pre-hair shaft; sg, second hair germ.</p

Wnt/β-catenin pathway is impaired in <i>Wls</i>-deficient skin.

<p>(A and B) Lef1 expression in the surface ectoderm is decreased, and no signal is detected in the underlying dermal mesenchyme (white arrows) in <i>Wls</i>-deficient skin. (C and D) Lef1 is detected both in ORS (white arrows) and IRS (yellow arrows) of control hair follicles. However the expression is indistinguishable in hair follicles of <i>K14-cre;Wls^c/c</i> skin. Abbreviations: epi, epidermis; der, dermis.</p