Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: FoxP1 is up‐regulated in multiple models of cancer cachexia in a FoxO1‐dependent manner. (A) Changes in FoxP1 mRNA in mouse skeletal muscle in various experimental models of cancer cachexia, including the subcutaneous murine colon 26 adenocarcinoma (C26) and Lewis lung carcinoma (LLC) models, and the human pancreas‐liver (L3.6pl) xenograft models, in which human L3.6pl cells were injected into the flank or orthotopically (ortho) into the pancreas. Note that independent unpaired two‐tailed t ‐tests were performed for the different cachexia models. (B–F) FOXP1 protein expression in skeletal muscle of C26 tumour‐bearing and non‐tumour‐bearing (Sham) mice. In panel (C), total FOXP1 expression was quantified by summing the signal from FOXP1A, FOXP1B, and FOXP1C and normalizing to total protein. (G) Transfection of rat solei with a constitutively active FoxO1 expression plasmid (ca. FoxO1) is sufficient to increase FoxP1 transcription, in vivo , as highlighted by its ability to increase a luciferase reporter gene driven by the FoxP1 promoter (RLU = relative light unit). (H) FoxO1 knockdown in mouse tibialis anterior , through transduction of muscles with AAV9 vector encoding FoxO1‐shRNA (or scrambled‐shRNA), prevents the C26‐induced upregulation of FoxP1 mRNA. For panels (A)–(G), depending on data distribution, unpaired two‐tailed t ‐tests or Mann–Whitney tests were performed to test for statistical differences between groups. A one‐way analysis of variance (ANOVA) was conducted to test for statistical differences between means in panel (H). Data are reported as mean ± SEM.

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Injection, Two Tailed Test, Expressing, Transfection, Plasmid Preparation, In Vivo, Luciferase, Knockdown, Transduction, Muscles, shRNA, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: Inducible, skeletal muscle‐specific FoxP1 over‐expression induces body and skeletal muscle wasting. FoxP1 over‐expression was induced by injecting mice intraperitoneally with tamoxifen for five consecutive days, followed by maintenance on a tamoxifen diet until study endpoint. Mice were euthanized following 6 weeks of treatment, or upon reaching IACUC‐mandated endpoint, based on cachexia development. (A–C) Tamoxifen treatment (started at 9–15 weeks of age) increases FoxP1 mRNA (A) and protein (B–C) expression in CRE+ FoxP1 iSkmTg/Tg mice compared with controls. (D–I) Skeletal muscle‐specific FoxP1 over‐expression induces body wasting (D–F) and skeletal muscle wasting (G–I). TAM = tamoxifen. (J) Representative images of tibialis anterior cross‐sections stained for wheat germ agglutinin (WGA), myosin heavy chain type I (MHCI), and myosin heavy chain type IIA (MHCIIA). CON = genetic controls. (K) Quantification of tibialis anterior muscle fibre cross‐sectional area (CSA) shows a leftward shift in fibre sizes in response to FoxP1 over‐expression. CSA data were binned and fit with a Gaussian least squares regression. Significance was determined by calculating the extra sum‐of‐squares F test. (L–M) Quantification of type IIA (L) and type IIX/B (M) fibre percentage in genetic control (CON) and CRE+ FoxP1 iSkmTg/Tg tibialis anterior . (N–O) Quantification of type IIA (N) and type IIX/B (O) muscle fibre CSA showing type IIX/B fibre atrophy in tibialis anterior muscles over‐expressing FoxP1. Statistical differences were determined by conducting unpaired two‐tailed t ‐tests or Mann–Whitney tests in panels (A) and (L–O), one‐way ANOVA in panel (C) and two‐way ANOVAs in panels (D)–(I). Data shown are from female mice and are reported as mean ± SEM. Note that data from male CRE+ FoxP1 iSkmTg/Tg mice and CRE‐ littermate controls are presented in Figure S3 .

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Over Expression, Expressing, Staining, Control, Muscles, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: Skeletal muscles of FoxP1 iSkmTg/Tg exhibit myopathy. (A) Representative images of haematoxylin and eosin (H&E) stained cross‐sections show increased extracellular space, presence of mononucleated cells, and increased number of myofibres with centralized nuclei in muscles from CRE+ FoxP1 iSkmTg/Tg mice compared with CRE‐ littermate controls (CON). (B) Quantification of total number of fibres presenting internalized nuclei in soleus muscle cross‐sections from CRE+ FoxP1 iSkmTg/Tg mice and CRE‐ littermate controls. (C) Representative muscle cross‐sections from female CRE+ FoxP1 iSkmTg/Tg mice and CRE‐ littermate controls stained with mouse immunoglobulin G (IgG) antibodies and wheat germ agglutinin (WGA). (D–F) muscles from female CRE+ FoxP1 iSkmTg/Tg mice show increased presence of intra‐myofibre endogenous IgG (D) and WGA (E) indicative of muscle damage, and increased percent area positive for WGA (F) compared to CRE‐ littermate controls. Data are reported as mean ± SEM. (G) Skeletal muscle cross‐sections from female CRE+ FoxP1 iSkmTg/Tg mice stained with Alizarin Red S show dysregulation of intracellular Ca 2+ compared with CRE‐ littermate controls. (H) Representative images of tibialis anterior cross‐sections stained with periodic acid‐Schiff (PAS) reveal the accumulation of PAS + vacuoles in muscles of CRE+ FoxP1 iSkmTg/Tg mice compared with CRE‐ littermate controls (CON). Depending on data distribution, unpaired two‐tailed t ‐tests or Mann–Whitney tests were performed to test for statistical differences between groups. Data are reported as mean ± SEM. Note that data from male CRE+ FoxP1 iSkmTg/Tg mice and CRE‐ littermate controls are presented in Figure S6 .

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Muscles, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: Inducible, skeletal muscle‐specific FoxP1 over‐expression causes ultra‐structural alterations. Transmission electron microscopy images show ultra‐structural alterations in muscles from female CRE+ FoxP1 iSkmTg/Tg mice compared with CRE‐ littermate controls. C, core; h, hypercontracted sarcomeres; L, lysosome; m. irr., membrane irregularities; N, internalized nucleus; NC, non‐contractile material; nf, necrotioc fibre; V, vacuoles.

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Over Expression, Transmission Assay, Electron Microscopy, Muscles, Membrane

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: Inducible, skeletal muscle‐specific FoxP1 over‐expression causes skeletal muscle weakness. Specific twitch (A) and tetanic (B) forces recorded from diaphragm strips from CRE+ FoxP1 iSkmTg/Tg mice (orange) and CRE‐ littermate controls (blue). Twitch contraction (C) and half relaxation times (D) show slower kinetics in female CRE+ FoxP1 iSkmTg/Tg mice vs. CRE‐ littermate controls. (E–F) Force–frequency relationships obtained from CRE+ FoxP1 iSkmTg/Tg mice and CRE‐ littermate controls indicate that diaphragms from CRE+ FoxP1 iSkmTg/Tg mice show reduced specific forces, and a leftward shift at low stimulation frequencies (F). Twitch (G) and tetanus (H) forces recorded from soleus muscles harvested from CRE+ FoxP1 iSkmTg/Tg mice (orange) and CRE‐ littermate controls. Depending on data distribution, unpaired two‐tailed t ‐tests or Mann–Whitney tests were performed to test for statistical differences between groups in panels (A–D) and (G–H). Statistical differences between means were tested by conducting two‐way mixed ANOVAs for panels (E) and (F). Data are reported as mean ± SEM.

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Over Expression, Muscles, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: Acute FoxP1 over‐expression in skeletal muscle leads to dysregulation of skeletal muscle homeostatic pathways. Mouse tibialis anterior muscles were transfected with a FoxP1 plasmid or empty vector and microarray analyses were conducted on pooled samples ( n = 3 per group). (A‐B) IPA enriched diseases and functions that are up‐regulated (A) and down‐regulated (B) in response to acute FoxP1 over‐expression. (C) RT‐qPCR validation for selected genes involved in muscle structural development, function, and maintenance. Depending on data distribution, unpaired two‐tailed t ‐tests or Mann–Whitney tests were performed to test for statistical differences between groups. Data are reported as mean ± SEM.

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Over Expression, Muscles, Transfection, Plasmid Preparation, Microarray, Quantitative RT-PCR, Biomarker Discovery, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: Inducible, skeletal muscle‐specific up‐regulation of FoxP1 impedes muscle regeneration following injury. (A–B) FoxP1 (A) and Acta1 (B) mRNA expression measured in proliferating C2C12 myoblasts (Day 0) and following 1, 2, or 5 days of differentiation into myotubes. (C–G) Tibialis anterior muscles of CRE+ FoxP1 iSkmTg/Tg mice and CRE‐ littermate controls (CON) were injured via direct injection of cardiotoxin on the same day as tamoxifen treatments were initiated. Twenty‐four days post‐injury, FoxP1 iSkmTg/Tg mice showed reduced muscle mass (C), and reduced cross‐sectional area (CSA) of regenerating myofibres, as visualized in tibialis anterior muscle cross‐sections stained with H&E (D) and as quantified in (E–G). Cross‐sectional area data were binned, fit with a Gaussian least squares regression and significance was determined by calculating the extra sum‐of‐squares F test. Statistical differences were determined by conducting one‐way ANOVAs in panels (A) and (B), and unpaired two‐tailed t ‐tests or Mann–Whitney tests in panels (C) and (E). Data are reported as mean ± SEM.

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Expressing, Muscles, Injection, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: FoxP1‐dependent induction of body and skeletal muscle wasting occurs through an HDAC‐dependent manner. (A–I) Tamoxifen‐treated CRE+ FoxP1 iSkmTg/Tg mice received daily intraperitoneal (IP) injections of trichostatin A (TSA, a pan HDAC inhibitor). TSA treatment blunted the FoxP1‐dependent loss of body mass (A) and skeletal muscle mass (B–E). Body and muscle masses are expressed as percent of sex‐matched CRE‐ littermate controls (CON). (F–G) Representative images of FoxP1 iSkmTg/Tg muscle cross‐sections stained with haematoxylin & eosin (H&E, F) and wheat germ agglutinin (WGA, G) show that TSA treatment protects against FoxP1‐induced muscle fibre atrophy (H), myopathy including degeneration/regeneration (I), and extracellular matrix (ECM) deposition (J). Data are normalized to CRE‐ littermate controls and reported as mean ± SEM. Cross‐sectional area data were binned, fit with a Gaussian least squares regression and significance was determined by calculating the extra sum‐of‐squares F test. Depending on data distribution, unpaired two‐tailed t ‐tests or Mann–Whitney tests were performed to test for statistical differences between groups in panels (A–E) and (I and J). [Note that data for mice not treated with TSA originate from the same mice as in Figures , , S3 , and S6 ].

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: FoxP1 mediates cancer‐induced muscle atrophy and the repression of MEF2 target genes. (A) Transduction of tibialis anterior muscles of C26‐tumour bearing mice with an AAV9 vector encoding FoxP1‐shRNA blunts the C26‐induced reduction in muscle fibre cross‐sectional area (CSA) that is observed in muscles transduced with AAV9 encoding scrambled‐shRNA. (B) Representative transmission electron microscopy images of tibialis anterior muscles from sham and C26‐tumour bearing mice transduced with the AAV9 encoding FoxP1 shRNA or scrambled shRNA, showing ultrastructural alterations in response to C26 tumour burden, that are improved in response to FoxP1 knockdown. (C) Microarray analyses conducted on pooled samples ( n = 3 per group) and further analysed with string to identify enriched cellular components, indicate that FoxP1 knockdown attenuates the tumour‐induced repression of genes involved in muscle structure/function (blue categories) and reduces the activation of genes involved in protein breakdown (red categories). (D–E) Genes repressed by two‐fold or more in response to C26 tumour burden that were rescued by two‐fold or more in muscles expressing FoxP1‐shRNA (249 genes) were further analysed using DAVID to identify enriched non‐redundant functional annotations (D), and using gene set enrichment analysis (GSEA) and the molecular signatures database to identify enriched transcription factor binding motifs among gene promoters (E). FoxP1 targets repressed in muscle of C26 mice were enriched for MEF2 and MYOD target genes involved in muscle contraction and muscle cell differentiation. (F) RT‐qPCR validation demonstrating that FoxP1 knockdown rescues the C26‐induced repression of several MEF2 target genes involved in muscle structural development, function, and maintenance. (G) Mouse tibialis anterior muscles were transfected with a FoxP1 expression plasmid or empty vector, plus a MEF2‐dependent luciferase reporter to assess MEF2 activity. RLU = relative light unit. Statistical differences were determined by conducting a one‐way ANOVA in panel (A), unpaired two‐tailed t ‐test or Mann–Whitney tests depending on data distribution in panel (F) and Wilcoxon test in panel (G). Data are reported as mean ± SEM.

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Transduction, Muscles, Plasmid Preparation, shRNA, Transmission Assay, Electron Microscopy, Knockdown, Microarray, Activation Assay, Expressing, Functional Assay, Binding Assay, Cell Differentiation, Quantitative RT-PCR, Biomarker Discovery, Transfection, Luciferase, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness

doi: 10.1002/jcsm.12666

Figure Lengend Snippet: FoxP1 expression is increased in skeletal muscle of patients with pancreatic ductal adenocarcinoma (PDAC) exhibiting cachexia. (A–B) FOXP1 protein expression is increased in rectus abdominis muscle biopsies from cachectic PDAC patients compared with non‐cancer control patients. In panel (A), arrows point to the two FoxP1 isoforms quantified in B. (C–E) FOXP1 mRNA expression in rectus abdominis muscle of PDAC patients correlates positively with body mass loss (C) and negatively with skeletal muscle index (D) and is increased in cachectic PDAC patients when compared with weight‐stable non‐cancer controls with normal muscularity (E). Note: mRNA data were extracted from microarray analyses previously analysed and published in combination with histological assessment of muscle biopsies ²⁰ and in combination with MRI‐based measures of skeletal muscularity and muscle quality. ⁶⁸ Statistical differences were determined by conducting unpaired two‐tailed t ‐test or Mann–Whitney tests depending on data distribution in panels (B) and (E), and simple linear regression analyses in panels (C) and (D). Data are reported as mean ± SEM.

Article Snippet: The FoxP1 promoter reporter plasmid (MPRM17622‐PG02) was purchased from Genecopoeia (Rockville, Maryland, USA).

Techniques: Expressing, Control, Microarray, Two Tailed Test, MANN-WHITNEY

Journal: Genes & Development

Article Title: Independent control of neurogenesis and dorsoventral patterning by NKX2-2

doi: 10.1101/gad.352886.125

Figure Lengend Snippet: In vitro generation of limb-level mouse motor neurons. ( A ) Schematic of ventral progenitor domains in the brachial (caudal cervical) spinal cord; at this level, p3 progenitors generate V3 interneurons (INs) instead of branchiovisceral motor neurons. ( B ) Summary of culture conditions that produce brachial motor neurons. ( C ) Immunofluorescence images of day 7 mouse cultures show that HOXC8 + somatic motor neurons (MNX1 + /ISL1 + ) are produced, some of which are FOXP1 + , indicating LMC-like identity. ( D ) Immunofluorescence images of day 5 cultures show that motor neurons (ISL1 + ) produced under these conditions are somatic (MNX1 + ). No expression of PHOX2A is observed. ( E ) RT-qPCR for Sim1 mRNA shows a >1000-fold expression increase in late stages of culture. ( F ) Summary of proportions of cells expressing NKX2-2, ISL1, and OLIG2 (normalized to the total number of cells expressing at least one of these three genes) over time.

Article Snippet: Primary antibodies, host species, and concentrations used in this study were as follows: ISL1 (goat, 1:5000, has 10% cross-reactivity to ISL2; Neuromics GT15051-100; RRID: AB_2126323), MNX1 (guinea pig, 1:100; from Jessell Laboratory), FOXP1 (mouse, 1:400; Santa Cruz Biotechnology sc-398811), NKX2-2 (mouse, 1:100; Developmental Studies Hybridoma Bank [DSHB] 74.5A5; RRID: AB_531794), BrdU (rat, 1:400; Abcam ab6326; RRID: AB_305426), OLIG2 (guinea pig, 1:100; from Jessell Laboratory), IRX3 (rabbit, 1:100; from Jessell Laboratory), and PAX6 (mouse, 1:100; DSHB supernatant).

Techniques: In Vitro, Immunofluorescence, Produced, Expressing, Quantitative RT-PCR

Journal: The Journal of Neuroscience

Article Title: Reduced Expression of Foxp1 as a Contributing Factor in Huntington's Disease

doi: 10.1523/jneurosci.3612-16.2017

Figure Lengend Snippet: Figure 1. Foxp1 expression levels in different brain regions. A, Foxp1 protein ϲϮϳ

Article Snippet: Flag-tagged Foxp1 plasmid, pCMV10-mFoxp1 was a gift from Benjamin Blencowe (Addgene plasmid #35170).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Reduced Expression of Foxp1 as a Contributing Factor in Huntington's Disease

doi: 10.1523/jneurosci.3612-16.2017

Figure Lengend Snippet: Figure 4. FOXP1 mRNA and protein expression in caudate of HD patients. ϲϲϭ

Article Snippet: Flag-tagged Foxp1 plasmid, pCMV10-mFoxp1 was a gift from Benjamin Blencowe (Addgene plasmid #35170).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Reduced Expression of Foxp1 as a Contributing Factor in Huntington's Disease

doi: 10.1523/jneurosci.3612-16.2017

Figure Lengend Snippet: Figure 6. Overexpression of Foxp1 protects neurons from apoptosis. A, ϲϵϲ

Article Snippet: Flag-tagged Foxp1 plasmid, pCMV10-mFoxp1 was a gift from Benjamin Blencowe (Addgene plasmid #35170).

Techniques: Over Expression

Journal: The Journal of Neuroscience

Article Title: Reduced Expression of Foxp1 as a Contributing Factor in Huntington's Disease

doi: 10.1523/jneurosci.3612-16.2017

Figure Lengend Snippet: Figure 7. Foxp1 induces the expression of p21. A, N2A cells were transfected with ϳϭϲ

Article Snippet: Flag-tagged Foxp1 plasmid, pCMV10-mFoxp1 was a gift from Benjamin Blencowe (Addgene plasmid #35170).

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: Repression of Dlx1/2 Signaling by Nolz-1/Znf503 is Essential for Parcellation of the Striatal Complex into Dorsal and Ventral Striatum

doi: 10.1101/463398

Figure Lengend Snippet: (a, c) BrdU was pulse-labeled at E12.5 and then analyzed at E18.5. Foxp1 + area was used to define the total striatal complex area (dorsal and ventral striatum). The line between the septoeminential sulcus and the piriform cortex was used to separate the dorsal from ventral striatum. The green round signals are counted as BrdU + cells (insets). BrdU E12.5 cells were decreased in dorsal Nolz-1 KO striatum. In contrast, BrdU E12.5 cells were markedly increased in the enlarged olfactory tubercle (OTe) of Nolz-1 KO ventral striatum. (b, d) Similar to the distribution of BrdU E12.5 cells, BrdU E15.5 cells were decreased in dorsal striatum, but increased in ventral striatum of Nolz-1 KO striatum. The total numbers of BrdU E12.5 cells (c) or BrdU E15.5 cells (d) are not changed in Nolz-1 KO striatum. *, p < 0.05; **, p < 0.01. n = 3/group. Scale bars in (a, b) , 200 μm.

Article Snippet: A.M. Graybiel of MIT), polyclonal goat anti-D1R antibody (1:2,000, Frontier Institute, Japan), polyclonal goat anti-D2R antibody (1:2,000, Frontier Institute, Japan), polyclonal rabbit anti-DARPP32 (1:2000; Cell Signaling), monoclonal mouse anti-Foxp1 antibody (1:1,000; kindly provided by Dr. J. Cordell of John Radcliffe Hospital, Oxford, UK), polyclonal rabbit anti-Foxp2 antibody (1:1,000; Abcam), polyclonal chicken anti-Green Fluorescent Protein (GFP; 1:1000; Abcam), polyclonal rabbit anti-Isl1 antibody (1:1,000, Abcam), monoclonal 3A4 or 4D5 mouse anti-Isl1 antibody (1:100, Developmental Studies Hybridoma Bank), monoclonal mouse anti-Ki67 (1:500 BD Pharmingen TM ), polyclonal rabbit anti-met-enkephalin antibody (1:5,000, ImmunoStar), polyclonal rabbit anti-Nolz-1 antibody (1:1,000) , monoclonal rabbit anti-Notch1 antibody (1:1,000, Abcam), polyclonal rabbit anti-Neuropilin-2 antibody (1:2,000, Cell Signaling), polyclonal goat anti-Plexin D1 antibody (1:1,000, R&D system), polyclonal rabbit anti-phospho-Histone H3 antibody (1:1,000, Millipore), monoclonal rabbit anti-Sox1 antibody (1:200; Epitomics), polyclonal rabbit anti-substance P antibody (1:3,000, Eugene Tech Inc.).

Techniques: Labeling

Journal: Cell Death & Disease

Article Title: Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy

doi: 10.1038/s41419-018-1081-0

Figure Lengend Snippet: Representative images illustrating the presence of a HOXB4 + and b HOXC8 + cells in spinal organoids. Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. c Co-staining of FOXP1 (green) and ISL1 (red) demonstrates presence of limb-innervating neurons in spinal organoids. Scale bars, 100 μm. d Representative images of spinal organoids at day 42 stained with ISL1 (red) and ChAT (green). Cellular nuclei were counterstained with DAPI. Scale bars, 100 μm. Spinal organoids are stained with e CHX10 + cells (RED) and f CALB + cells (green). Scale bars, 100 μm. g Co-staining of S100β and TUJ1 shows presence of astrocytes in spinal organoids. Scale bars, 100 μm. h Quantitative-PCR analysis demonstrates a lack of dorsal cell types in the spinal organoids generated

Article Snippet: The following primary antibodies (and their respective dilutions) were used: rabbit SOX1 (1:1000) (Abcam, ab87775), mouse Nestin (1:1000) (Abcam, ab22035), rabbit ISL1 (1:1500) (Abcam, ab109517), rabbit cleaved Caspase-3 (1:1000) (Cell Signaling Technology, #9661), mouse Ki67 (1:1500) (Cell Signaling Technology, #9449), mouse SMI-32 (1:1000) (Calbiochem, NE-1023), mouse SMN (1:400) (BD Pharmingen, 610647), rabbit Ki67 (1:250) (Abcam, ab16667), mouse TUJ1 (1:2000) (Biolegend, #801202), goat SOX10 (1:100) (Santa Cruz Biotechnologies, sc-17342), rabbit HOXB4 (1:200) (Abcam, ab133521), rabbit HOXC8 (1:200) (Abcam, ab86236), rabbit Calbindin (1:1000) (Abcam, ab11426), mouse FoxP1 (1:100) (R&D Systems, MAB45341), and sheep Chx10 (1:200) (Abcam, ab16141).

Techniques: Staining, Real-time Polymerase Chain Reaction, Generated