N-RasG12V transformation requires the MEK-ERK pathway and S727 phosphorylation of mitochondrial STAT3.

<p>N-RasV12 transformed wild-type (NRas), STAT3-/- MEFs (NRas:STAT3-/-) or NRas:STAT3-/- MEFs stably transduced with mitochondrially restricted STAT3 (NRas: STAT3-/- + MTS-STAT3) or mitochondrially restricted phospho-S727 mimetic mutant STAT3 (NRas: STAT3-/- + MTS-STAT3 S/D) cells were plated in media supplemented with 0.3% agar and the indicated dose of PD0325901 or vehicle (unt) and colonies counted 14 d later. Each point represents the mean of biological triplicates, error bars are ±1 standard deviation from the mean. We used Student’s t-test to determine significant differences.* denotes p<0.05.</p

H-RasV12 transformation requires the MEK-ERK pathway and S727 phosphorylation of mitochondrial STAT3.

<p>(<b>A</b>) STAT3^-/- MEFs were stably transduced with H-RasV12 and empty vector (EV) or wild-type STAT3 (WT STAT3). STAT3^-/- MEFs were also transduced with H-RasV12 effector domain mutants (35S to activate the MEK-ERK pathway and 40C to activate the PI3K pathway) in addition to a mitochondrially restricted STAT3 (35S or 40C+MTS STAT3) or a mitochondrially restricted phospho-S727 mimetic STAT3 (35S or 40C+MTS STAT3 S/D). 10³ cells were plated in media supplemented with 0.3% agar and colonies counted 14 d later. (<b>B</b>) Wild type MEFs were stably transduced with H-RasV12, H-Ras35S or H-Ras40C and 10³ cells were plated in media supplemented with 0.3% agar and colonies counted 14 d later. (C) STAT3^-/- MEFs were stably transduced with H-RasV12 and wild-type STAT3 (H-RasV12+STAT3) empty vector (H-RasV12+EV), S727A STAT3 (H-RasV12+SA) or S727D STAT3 (H-RasV12+SD). 10³ cells were plated in media supplemented with 0.3% agar and colonies counted 14 d later. (<b>D</b>) H-RasV12 transformed wild-type (H-Ras:STAT3+/+), STAT3-/- MEFs (H-Ras:STAT3-/-) or H-Ras:STAT3-/- MEFs stably transduced with mitochondrially restricted STAT3 (H-Ras:MTS-STAT3) or mitochondrially restricted phospho-S727 mimetic mutant STAT3 (H-Ras:MTS-STAT3 S/D) cells were plated in media supplemented with 0.3% agar and the indicated dose of PD0325901 or vehicle (unt) and colonies counted 14 d later. Each point represents the mean of biological triplicates, error bars are ±1 standard deviation from the mean. Student’s t-test was used to determine significant differences. * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.0001. </p

H-Ras phosphorylates STAT3 on S727 via the MEK-ERK pathway.

<p>(<b>A</b>) Protein extracts from MEFs transformed with H-RasV12 (V12) or H-RasV12 effector domain mutants (35S or 40C) were resolved by 10% SDS-PAGE, transferred to PVDF membranes and probed with phospho-specific antibodies against pY705 STAT3 (pY STAT3), pS727 STAT3 (pS STAT3), pY204 ERK (pERK), pS473 Akt (pAKT), or total STAT3, ERK, AKT and tubulin, as indicated. Protein lysate from mouse liver 4 h following an injection with 30µg of lipopolysaccharide was used as a positive control (+ve). (<b>B</b>) H-RasV12 transformed MEFs were treated with DMSO (unt), Rapamycin (10nM), LY294002 (50µM) or PD0325901 (1µM) for 2 h. Cell lysates were processed for immunoblotting and probed with phospho-specific antibodies against pS727 STAT3 (pS STAT3), pT389 p70 S6 Kinase, pY204 ERK (pERK), pS473 Akt (pAKT), or total STAT3 and tubulin as loading controls. (<b>C</b>) H-RasV12 transformed MEFs were treated with the indicated dose of PD0325901 or vehicle (DMSO) for 2 h. Cell lysates were processed for immunoblotting and probed with phospho-specific antibodies against pS727 STAT3 (pS STAT3), pY204 ERK (pERK), pS473 Akt (pAKT), or total STAT3 and total ERK as loading controls. (<b>D</b>) N-Ras transformed human THP-1 acute myelocytic leukemia cells were treated with the indicated doses of PD0325901 (nM) or vehicle (DMSO) for 2 h prior to the preparation of cell lysates for immunoblotting. Lysates were probed for the indicated proteins and phospho-proteins as described in panel (C).</p

K-RasG12V transformation requires the MEK-ERK pathway and S727 phosphorylation of mitochondrial STAT3.

<p>K-RasV12 transformed wild-type (KRas), STAT3-/- MEFs (KRas:STAT3-/-) or KRas:STAT3-/- MEFs stably transduced with mitochondrially restricted STAT3 (KRas:STAT3-/-+MTS-STAT3) or mitochondrially restricted phospho-S727 mimetic mutant STAT3 (KRas: STAT3-/- +MTS-STAT3 S/D) cells were plated in media supplemented with 0.3% agar and the indicated dose of PD0325901 or vehicle (unt) and colonies counted 14 d later. Each point represents the mean of biological triplicates, error bars are ±1 standard deviation from the mean. We used Student’s t-test to determine significant differences. * denotes p<0.05, *** denotes p<0.001. </p

Percentages of MRs showing phenotypic abnormalities in female <i>Gli2^1ki/1ki; Gli3^xt/+</i> embryos at E18.5.

<p>Numbers represent percentages of MRs showing loss, evagination or impairment in invagination from a total ‘n’. Abbreviations: Imp. Invag: Impaired Invagination; n: total number of MRs analyzed.</p

Expression and modulation of <i>Gli1-LacZ</i> in the adult nipple during the pregnancy cycle.

<p>Gli1-LacZ expression is visible at the neck of the mammary sprout (red arrow) in skin whole-mounts of E18.5 <i>Gli1^lzki/+</i> embryos at low (A) and higher power (A’). Histological section through the sprout shows expression of Gli1-LacZ within the stroma (red arrow) surrounding the sprout (black arrow) underneath the nipple sheath (black arrowheads) at low (B) and higher power (B’). <i>Gli1-lacZ</i> is expressed within the dermal component but not the epithelium in virgin (C), mid pregnant (D), lactating (E) and involuting (F) nipples. Immunohistochemistry for desmin (G, J), SMA (H, K) and vimentin (I, L) on serial sections of a 15.5 day pregnant nipple demonstrated that <i>Gli1-lacZ</i> was expressed in both smooth muscle cells and fibroblasts but not myofibroblasts. <i>Gli1-lacZ</i> was also found near and surrounding peripherin positive nerve tracts (M) and both Von Willebrand positive vessels (N).</p

Evagination of MR#2 and encroachment of hair follicles in <i>Gli3</i>^<i>xt</i>/xt embryos.

<p>Analysis of outer surface of E18.5 skin whole-mounts (A–D) stained with carmine (C, D) and sections stained with nuclear fast red (E, F) and X-Gal (blue) to detect hair follicles expressing the Gli1-LacZ reporter (A–F). MR#1 (A) from <i>Gli3^xt/xt; Gli1^lzki/+</i> mutants and MR#2 from control <i>Gli1^lzki/+</i> (C) embryos show normal invagination and appropriate exclusion of hair follicles. In contrast MR#2 from <i>Gli3^xt/xt; Gli1^lzki/+</i> mutants (B, D) showed prominent evagination and encroachment of hair follicles.</p

Percentages of evaginated MRs in male <i>Gli3^xt/xt</i> embryos at E16.5 and E18.5.

<p>Numbers represent percentages of MRs that protrude from the surface of male skin whole-mounts from a total ‘n’. Abbreviations: E: embryonic day; n: total number of putative sites for MRs on male skin whole mounts.</p

Percentages of MRs showing retention in male <i>Gli3^xt/xt</i> embryos at E16.5 and E18.5.

<p>Numbers represent percentages of MRs retained on male skin whole-mounts from a total ‘n’. Abbreviations: E: embryonic day; n: total number of putative sites for MRs on male skin whole mounts.</p

Misactivation of Hh signaling detrimentally affects MR invagination and hair follicle suppression in females and eradication of MRs in males.

<p>X-Gal stained whole-mounts (A–E) and sections (F-H) of <i>Gli2^1ki/1ki;Gli3^xt/+;Gli1^lzki/+</i> embryos were examined at E18.5. In whole-mounts of female skins, MR#2 protruded prominently and showed encroachment of hair follicles inappropriately close to the evaginated MR (A). Examination of male skin whole-mounts revealed retention of MR#1, #2 and #4 at low (B) and high power (C, D, E respectively), similar to that seen in <i>Gli3^xt/xt;Gli1^lzki/+</i> embryos. Serial sections through a male MR#2 from <i>Gli2^1ki/1ki;Gli3^xt/+;Gli1^lzki/+</i> embryos showed Gli1-LacZ-positive hair follicles close to the protruding bud (F; NFR counterstain) and weak expression of Tenascin C (G) and loss of AR (H) by immunohistochemistry.</p