Journal: Oncogene

Article Title: Regulation of IMP3 by EGFR Signaling and Repression by ERβ: Implications for Triple Negative Breast Cancer

doi: 10.1038/onc.2011.620

Figure Lengend Snippet: (A) Immunoblots show the effect of blocking EGFR on IMP3 expression using a specific Ab (2 μg /mL) in MDA-MB-231 (left) and MDA-MB-468 (right) cells. Rabbit IgG was used as control. (B) & (C) MDA-MB-231 and MDA-MB-468 cells were treated with MEK1/2 inhibitors u0126 (10 μM) and PD98059 (50 μM) for different time points as indicated in the figure, and the expression of IMP3, as well as pMAPK, was analyzed by immunoblotting. IMP3 mRNA from cells treated with U0126 for 10 hr was assessed by qPCR. (D) MDA-MB-231 cells were transfected with a luciferase reporter construct containing 2.872 kb IMP3 promoter (wild type) in presence or absence of U0126 and PD98059, and luciferase activity was measured 24 h post-transfection. Data represent the mean of three independent experiments. p value (*) < 0.05.

Article Snippet: The MEK1/2 inhibitors U0126 and PD98059 were purchased from LC Laboratories.

Techniques: Western Blot, Blocking Assay, Expressing, Transfection, Luciferase, Construct, Activity Assay

Journal: bioRxiv

Article Title: Differential effect of Fas activation on spinal muscular atrophy motoneuron death and induction of axonal growth

doi: 10.1101/2022.05.12.491475

Figure Lengend Snippet: Death of Smn 2B/- motoneurons occurs through the Fas death pathway. (a) Smn 2B/- motoneurons were maintained in culture in the presence (or not) of Fas-Fc (1 μg/ml) or sFasL (100 ng/ml in the presence of 1 μg/ml enhancer antibody). Motoneuron survival was determined after 2 DIV and expressed relative to the number of motoneurons at the time of plating (0 DIV) in each condition. (b) Motoneurons from E12.5 spinal cord of Hb9::GFP embryos were cultured for 24 h and then treated (or not) with sFasL. The percentage of GFP + FoxP1 - and GFP + FoxP1 + motoneurons killed by Fas was determined 48 h later. (c) Smn 2B/- motoneurons were cultured in the presence of Fas-Fc (1 μg/ml), z-IETD-fmk (10 μM), Ac-LEHD-cmk (1 μM), z-DEVD-fmk (10 μM) or SB203580 (10 μM). Motoneuron survival was determined after 3 DIV and expressed relative to the number of motoneurons at the time of plating (0 DIV, none). (d) Smn 2B/+ and Smn 2B/- motoneurons were cultured for 24 h and immunostained with anti-Islet-1/-2 and CRMP4 antibodies. Scale bar, 25 μm. (e) For the quantification of CRMP4 immunostaining, the CRMP4 mean fluorescence is expressed relative to Islet-1/-2 fluorescence (a.u, arbitrary unit). A total of 90 motoneurons were quantified by condition ( n = 3). (f) SMA motoneurons were infected with 10 TU per cell of AAV-shCrmp4 and AAV-shCrmp4 mis after plating. The survival of motoneurons was determined at 3 DIV, expressed relative to the control condition (none) at the time of seeding (0 DIV). (g) Smn 2B/- motoneurons were incubated (or not) with 10 μM of Y-27632 and 10 μM of PD98059 for 3 days. Motoneuron survival was expressed relative to the control condition at 0 DIV. Values are means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, n.s, non-significant, ANOVA with Tukey’s post hoc test (a,c,f,g) or t test (b,e). Data are representative of at least three independent experiments, each done in triplicate.

Article Snippet: Soluble recombinant human FasL, enhancer for ligand and recombinant human Fas-Fc were purchased from Enzo Life Sciences. z-IETD-fmk, z-DEVD-fmk, Ac-LEHD-cmk, SB203580, PD98059 and Y-27632 were purchased from Merck.

Techniques: Cell Culture, Immunostaining, Fluorescence, Infection, Control, Incubation

Journal: bioRxiv

Article Title: Differential effect of Fas activation on spinal muscular atrophy motoneuron death and induction of axonal growth

doi: 10.1101/2022.05.12.491475

Figure Lengend Snippet: Fas-induced axon outgrowth requires ERK and Caspase activity. (a) Hb9::GFP motoneurons were maintained in culture for 24 h and treated with sFasL (100 ng/ml and 1 μg/ml enhancer antibody) in combination with PD98059 (10 μM). Total axon length was calculated 24 h later. (b) Motoneurons were cultured for 24 h and treated with sFasL and PD98059. Surviving GFP + motoneurons were directly counted under fluorescence microscope 48 h later. (c , d) Twenty-four hours post seeding, sFasL (100 ng/ml) and Y-27632 (10 μM) were added to Hb9::GFP motoneurons. (c) Total axon length was determined 24 h later. (d) Motoneuron survival was determined 48 h later. (e) Motoneurons were maintained in culture for 24 h and treated with Fas activator and caspase-9 inhibitor Ac-LEHD-cmk (1 μM). Motoneuron survival was determined 48 h later and expressed relative to the non-treated condition. (f) Motoneurons were treated (or not) with sFasL in combination with Ac-LEHD-cmk (1 μM) or z-DEVD-fmk (10 μM). Axon length was determined 24 h later. The graphs show the mean values ± SEM of at least three independent experiments performed in triplicate, ** P < 0.01, *** P < 0.001, n.s, non-significant, ANOVA with Tukey’s post hoc test.

Article Snippet: Soluble recombinant human FasL, enhancer for ligand and recombinant human Fas-Fc were purchased from Enzo Life Sciences. z-IETD-fmk, z-DEVD-fmk, Ac-LEHD-cmk, SB203580, PD98059 and Y-27632 were purchased from Merck.

Techniques: Activity Assay, Cell Culture, Fluorescence, Microscopy