Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin fragments are differentially localized in neurons. ( A ) WB of mouse whole brain protein samples with menin C-terminal ((C); left panel) and N-terminal ((N); right panel) epitope antibodies (n = 3 each, representative blots), depicting full length menin (black arrow), as well as N-terminal (light grey arrow) and C-terminal (dark grey arrow) menin proteolytic fragments. ( B ) WB of subcellular fractions from mouse brain protein samples with menin C-terminal ((C); top and middle panels) and N-terminal ((N); bottom panel) epitope antibodies (n = 6, representative blots). N denotes nuclear fraction, C denotes cytoplasmic fraction, S denotes synaptic fraction. Menin localizes to the nucleus, the C-menin fragment localizes to synaptic membranes, and the N-menin fragment localizes to the cytoplasm. ( C ) As in ( B ), the markers histone H3 (HH3; nuclear marker, top panel), β-tubulin (TUB; cytoplasmic marker, middle panel), and synaptophysin (SYP; synaptic marker, bottom panel), are shown to verify the subcellular fractions. ( D ) ICC localization of menin in hippocampal cultures at DIV 7 (n = 13 images, 4 independent samples, representative image), with N-terminal (i) and C-terminal (ii) epitope antibodies, and the nuclear stain DAPI (iii), (iv) shows merged channels. α-N-terminal menin signals are restricted to the nuclear and perinuclear region, α-C-terminal menin signals also exhibit punctate localization along neurites. Scale bar, 50 μm. See also Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Marker, Staining

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin is cleaved by calpain. ( A ) ICC localization of menin in hippocampal cultures at DIV 7, cultured in the presence of 0.1% DMSO vehicle control (i–v) or 20 μM PD150606 (vi–x), a cell permeable calpain inhibitor (n = 9 images, 3 independent samples each, representative images). Cells were labeled with the nuclear stain DAPI (i,vi), menin N-terminal (ii,vii) and C-terminal (iii,viii) epitope antibodies, and a neurofilament antibody (iv,ix), (v,x) shows merged channels. Scale bar, 50 μm. ( B ) Enlarged region of interest (ROI) depicting neurite structure from the boxed region shown in Av (i–iii) and Ax (iv–vi). Scale bar, 10 μm. ( C , D ) Summary data, fluorescence intensity of the nuclear α-N-menin signal was unaffected ( C ; ROIs: 0.1% DMSO n = 67, 20 μM PD150606 n = 59), whereas the neurite α-C-menin signal was reduced upon calpain inhibition ( D ; ROIs: n = 27 each). ***Statistical significance (independent t-test), P < 0.001. See also Table and Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Cell Culture, Labeling, Staining, Fluorescence, Inhibition

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nAChR α5 subunit expression. ( A ) ICC characterization of menin and nAChR protein expression in neuronal soma from NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures (n ≥ 4 images, ≥2 independent samples; see Table ; representative images, DIV 7). Untransduced neurons were GFP negative (−) and transduced neurons were GFP positive (+). The expression of menin was determined with C-terminal (i,v) and N-terminal (ii,vi) epitope antibodies, and nAChRs with a nAChR α5 antibody (iii,vii), or fluorophore-tagged α-BTX (iv,viii; α7-nAChR). ICC labels are shown in a (left panels), and GFP fluorescence is shown in b (right panels). Scale bars, 20 μm. ( B–E ) Summary data, normalized somal fluorescence intensity of α-C-terminal menin ( B ), α-N-terminal menin ( C ), α-nAChR α5 ( D ), and α-BTX ( E ), in GFP + neurons relative to GFP- neurons at DIV 3–14 (ROIs: n ≥ 12 each; see Table ). Dashed lines represent a 1:1 ratio indicating no change. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Expressing, shRNA, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: C-menin mediates neurite localization of α7 subunit-containing nAChRs. ( A , B ) ICC characterization of the neurite localization of C-menin and α7-nAChRs in NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures ( A ; n = 18 images, 5 independent samples each; representative images, DIV 7) or hippocampal cultures maintained in the presence of 0.1% DMSO or 20 μM PD150606 ( B ; n = 12 images, 2 independent samples each; representative images, DIV 7). Cells were labeled with the menin C-terminal epitope antibody (i-ii-a) or fluorophore-tagged α-BTX (iii-iv-a), and neurites were visualized with GFP ( A i-iv-b) or neurofilament ( B i-iv-b) antibodies, (i-iv-c) shows merged channels. Scale bars, 20 μm. ( C , D ) Summary data, the α-C-menin signal in neurites was reduced upon MEN1 knockdown ( C ), which coincides with a reduction of the neurite α-BTX signal ( D ). ( E ) Summary data, reduction of C-menin abundance by calpain inhibition (see Fig. ) coincides with a reduction of the neurite α-BTX signal (ROIs: n ≥ 36 each; see Table ), indicating that the synaptic targeting of α7-nAChRs requires the menin C-terminal fragment. Asterisks, statistical significance (Mann-Whitney U test); * P < 0.05. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: shRNA, Labeling, Inhibition, MANN-WHITNEY