brn2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc brn2
    ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and <t>BRN2.</t> MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.
    Brn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Deletion of TRPC6 , an autism risk gene, induces hyperexcitability in cortical neurons derived from human pluripotent stem cells"

    Article Title: Deletion of TRPC6 , an autism risk gene, induces hyperexcitability in cortical neurons derived from human pluripotent stem cells

    Journal: bioRxiv

    doi: 10.1101/2022.11.14.516407

    ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and BRN2. MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.
    Figure Legend Snippet: ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and BRN2. MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.

    Techniques Used: Immunostaining, Clone Assay, Marker, Staining, Expressing

    brn2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc brn2
    ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and <t>BRN2.</t> MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.
    Brn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Deletion of TRPC6 , an autism risk gene, induces hyperexcitability in cortical neurons derived from human pluripotent stem cells"

    Article Title: Deletion of TRPC6 , an autism risk gene, induces hyperexcitability in cortical neurons derived from human pluripotent stem cells

    Journal: bioRxiv

    doi: 10.1101/2022.11.14.516407

    ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and BRN2. MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.
    Figure Legend Snippet: ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and BRN2. MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.

    Techniques Used: Immunostaining, Clone Assay, Marker, Staining, Expressing

    anti brn2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti brn2
    Differentiation of SMNs, cortical neurons, and sensory neurons using inducible transcription factors. (A) Distinct tet-inducible transcription factors contained within PiggyBac constructs were nucleofected into iPSCs to generate different cell types. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1 staining. Darker, lighter, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C) Cyclic immunofluorescence of SMNs for HB9, NEUN, ISL1, TUJ1, and two different CHAT antibodies; quantified in (D). “Combined” indicates the percentage of cells positive for all markers. (E) Cortical neurons stained for <t>BRN2,</t> FOXG1, and TUJ1; quantified (F). (G) Sensory neurons stained for BRN3A, ISL1, and TUJ1; quantified in (H). Scale bars = 100uM; n=8 wells for all groups. Across panels, arrows indicate the same neuron, which is magnified in the inset.
    Anti Brn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "iPSC Motor Neurons with Familial ALS Mutations Capture Gene Expression Changes in Postmortem Sporadic ALS Motor Neurons"

    Article Title: iPSC Motor Neurons with Familial ALS Mutations Capture Gene Expression Changes in Postmortem Sporadic ALS Motor Neurons

    Journal: bioRxiv

    doi: 10.1101/2022.10.25.513780

    Differentiation of SMNs, cortical neurons, and sensory neurons using inducible transcription factors. (A) Distinct tet-inducible transcription factors contained within PiggyBac constructs were nucleofected into iPSCs to generate different cell types. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1 staining. Darker, lighter, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C) Cyclic immunofluorescence of SMNs for HB9, NEUN, ISL1, TUJ1, and two different CHAT antibodies; quantified in (D). “Combined” indicates the percentage of cells positive for all markers. (E) Cortical neurons stained for BRN2, FOXG1, and TUJ1; quantified (F). (G) Sensory neurons stained for BRN3A, ISL1, and TUJ1; quantified in (H). Scale bars = 100uM; n=8 wells for all groups. Across panels, arrows indicate the same neuron, which is magnified in the inset.
    Figure Legend Snippet: Differentiation of SMNs, cortical neurons, and sensory neurons using inducible transcription factors. (A) Distinct tet-inducible transcription factors contained within PiggyBac constructs were nucleofected into iPSCs to generate different cell types. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1 staining. Darker, lighter, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C) Cyclic immunofluorescence of SMNs for HB9, NEUN, ISL1, TUJ1, and two different CHAT antibodies; quantified in (D). “Combined” indicates the percentage of cells positive for all markers. (E) Cortical neurons stained for BRN2, FOXG1, and TUJ1; quantified (F). (G) Sensory neurons stained for BRN3A, ISL1, and TUJ1; quantified in (H). Scale bars = 100uM; n=8 wells for all groups. Across panels, arrows indicate the same neuron, which is magnified in the inset.

    Techniques Used: Construct, Staining, Immunofluorescence

    anti brn2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti brn2
    Anti Brn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tween 20 pbst  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc tween 20 pbst
    Tween 20 Pbst, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    primary antibodies brn2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc primary antibodies brn2
    NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C) . (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker <t>BRN2</t> (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G i ) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G ii ) . (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10 8 ) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I) , single stained with DiI (J) , and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.
    Primary Antibodies Brn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers"

    Article Title: Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers

    Journal: Frontiers in Integrative Neuroscience

    doi: 10.3389/fnint.2022.879832

    NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C) . (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker BRN2 (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G i ) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G ii ) . (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10 8 ) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I) , single stained with DiI (J) , and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.
    Figure Legend Snippet: NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C) . (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker BRN2 (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G i ) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G ii ) . (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10 8 ) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I) , single stained with DiI (J) , and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.

    Techniques Used: Marker, Flow Cytometry, Staining, Labeling, Derivative Assay, Cell Culture, Immunostaining, Microscopy, Western Blot, Isolation

    anti brn2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti brn2 antibody
    Anti Brn2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    brn2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc brn2
    ( A and B ) Dynamic expressions of PAX6 and <t>BRN2</t> during the monkey cortical expansion through brain coronal sections of the primary visual cortex. BRN2 was initially expressed in NEPCs at embryonic day 30 (E30) (A) and gradually extended along the VZ spread to SVZ at E49 (B). Cortical layers were identified by staining of 4′,6-diamidino-2-phenylindole (DAPI) and PAX6. ( C ) Immunohistochemical analyses for Brn2 and Pax6 were performed in E9.5 (top) and E12.5 (bottom) mouse neocortices. ( D ) Images showing the expression patterns of BRN2 in E40 monkey coronal brain sections of the primary visual cortex. ( E ) Double staining of BRN2 and PAX6 in the E40 VZ and SVZ of BRN2-expressing (BRN2 + ) or BRN2-absent (BRN2 − ) zones, respectively. An obvious SVZ was formed in the BRN2 + zone but not in the BRN2 − zone. ( F ) Quantification of BRN2 and PAX6 expression in the BRN2 + and BRN2 − zones, respectively. Data are presented as means ± SEM ( n = 3 slices); >300 cells per group were counted. ( G ) Expressions of p-VIMENTIN (PVI) and KI67 in the E40 VZ and SVZ of BRN2 + or BRN2 − zones, respectively. The quantification data were only from the cells in the apical regions where cell division occurs. Data are presented as means ± SEM, n = 3 slices; >400 cells per group were counted. Arrowheads indicate positive cells. (a to d) Areas in rectangles are shown with higher magnification. ( H ) The left panel is a cortical graph at E40, and the right panel is the schematic diagram for measuring the thicknesses of whole-cortex and BRN2-expressing cells in monkey fetal brain. ( I ) Coordinating BRN2 distribution with the thicknesses of the cortex in E40 monkey neocortex, relative to (H). Start and lateral sites in (H) correspond to those in (I). Blue, DAPI, nuclear staining. Scale bars, 100 μm.
    Brn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "BRN2 as a key gene drives the early primate telencephalon development"

    Article Title: BRN2 as a key gene drives the early primate telencephalon development

    Journal: Science Advances

    doi: 10.1126/sciadv.abl7263

    ( A and B ) Dynamic expressions of PAX6 and BRN2 during the monkey cortical expansion through brain coronal sections of the primary visual cortex. BRN2 was initially expressed in NEPCs at embryonic day 30 (E30) (A) and gradually extended along the VZ spread to SVZ at E49 (B). Cortical layers were identified by staining of 4′,6-diamidino-2-phenylindole (DAPI) and PAX6. ( C ) Immunohistochemical analyses for Brn2 and Pax6 were performed in E9.5 (top) and E12.5 (bottom) mouse neocortices. ( D ) Images showing the expression patterns of BRN2 in E40 monkey coronal brain sections of the primary visual cortex. ( E ) Double staining of BRN2 and PAX6 in the E40 VZ and SVZ of BRN2-expressing (BRN2 + ) or BRN2-absent (BRN2 − ) zones, respectively. An obvious SVZ was formed in the BRN2 + zone but not in the BRN2 − zone. ( F ) Quantification of BRN2 and PAX6 expression in the BRN2 + and BRN2 − zones, respectively. Data are presented as means ± SEM ( n = 3 slices); >300 cells per group were counted. ( G ) Expressions of p-VIMENTIN (PVI) and KI67 in the E40 VZ and SVZ of BRN2 + or BRN2 − zones, respectively. The quantification data were only from the cells in the apical regions where cell division occurs. Data are presented as means ± SEM, n = 3 slices; >400 cells per group were counted. Arrowheads indicate positive cells. (a to d) Areas in rectangles are shown with higher magnification. ( H ) The left panel is a cortical graph at E40, and the right panel is the schematic diagram for measuring the thicknesses of whole-cortex and BRN2-expressing cells in monkey fetal brain. ( I ) Coordinating BRN2 distribution with the thicknesses of the cortex in E40 monkey neocortex, relative to (H). Start and lateral sites in (H) correspond to those in (I). Blue, DAPI, nuclear staining. Scale bars, 100 μm.
    Figure Legend Snippet: ( A and B ) Dynamic expressions of PAX6 and BRN2 during the monkey cortical expansion through brain coronal sections of the primary visual cortex. BRN2 was initially expressed in NEPCs at embryonic day 30 (E30) (A) and gradually extended along the VZ spread to SVZ at E49 (B). Cortical layers were identified by staining of 4′,6-diamidino-2-phenylindole (DAPI) and PAX6. ( C ) Immunohistochemical analyses for Brn2 and Pax6 were performed in E9.5 (top) and E12.5 (bottom) mouse neocortices. ( D ) Images showing the expression patterns of BRN2 in E40 monkey coronal brain sections of the primary visual cortex. ( E ) Double staining of BRN2 and PAX6 in the E40 VZ and SVZ of BRN2-expressing (BRN2 + ) or BRN2-absent (BRN2 − ) zones, respectively. An obvious SVZ was formed in the BRN2 + zone but not in the BRN2 − zone. ( F ) Quantification of BRN2 and PAX6 expression in the BRN2 + and BRN2 − zones, respectively. Data are presented as means ± SEM ( n = 3 slices); >300 cells per group were counted. ( G ) Expressions of p-VIMENTIN (PVI) and KI67 in the E40 VZ and SVZ of BRN2 + or BRN2 − zones, respectively. The quantification data were only from the cells in the apical regions where cell division occurs. Data are presented as means ± SEM, n = 3 slices; >400 cells per group were counted. Arrowheads indicate positive cells. (a to d) Areas in rectangles are shown with higher magnification. ( H ) The left panel is a cortical graph at E40, and the right panel is the schematic diagram for measuring the thicknesses of whole-cortex and BRN2-expressing cells in monkey fetal brain. ( I ) Coordinating BRN2 distribution with the thicknesses of the cortex in E40 monkey neocortex, relative to (H). Start and lateral sites in (H) correspond to those in (I). Blue, DAPI, nuclear staining. Scale bars, 100 μm.

    Techniques Used: Staining, Immunohistochemical staining, Expressing, Double Staining

    ( A ) Workflow of generation and analysis of BRN2 -KO monkeys. RNP, gRNA-Cas9 nuclease complex. ( B ) Schematic diagram of target gene editing. Triangles with different colors show the different locations where the sgRNAs target. Two pairs of sgRNA (A+B or A+C) in principle induced type I [with about 1091–base pair (bp) deletion] and type II (with near-488-bp deletion) gene mutations, respectively. ( C ) Detailed information of all fetuses in this study. Two fetuses (080460 and 081012) were miscarried during gestation, and we failed to collect their tissues for further analysis. ( D ) Representative images of wild-type (WT; BRN2 +/+ ) and BRN2 -knockout (KO, BRN2 −/− ) fetus at E29, E36, and E49. Inserts are magnifications of monkey brains, showing abnormal cerebrovascular development in the BRN2 −/− E36 and E49 fetuses. Scale bars, 3.7 mm. NA, non-examination. ( E ) Agarose gel electrophoresis of the PCR products from all knockout monkey samples. BRN2 +/+ sample (123044-1#) is shown as a negative control of the target gene editing. P, placenta; B, brain cells. ( F ) Immunostainings of BRN2 and SOX1 in the BRN2 +/+ and BRN2 −/− monkey fetal cortex, confirming BRN2 deletion in the BRN2 −/− cortex. Blue, DAPI, nuclear staining. Scale bars, 100 μm. IHC, immunohistochemistry.
    Figure Legend Snippet: ( A ) Workflow of generation and analysis of BRN2 -KO monkeys. RNP, gRNA-Cas9 nuclease complex. ( B ) Schematic diagram of target gene editing. Triangles with different colors show the different locations where the sgRNAs target. Two pairs of sgRNA (A+B or A+C) in principle induced type I [with about 1091–base pair (bp) deletion] and type II (with near-488-bp deletion) gene mutations, respectively. ( C ) Detailed information of all fetuses in this study. Two fetuses (080460 and 081012) were miscarried during gestation, and we failed to collect their tissues for further analysis. ( D ) Representative images of wild-type (WT; BRN2 +/+ ) and BRN2 -knockout (KO, BRN2 −/− ) fetus at E29, E36, and E49. Inserts are magnifications of monkey brains, showing abnormal cerebrovascular development in the BRN2 −/− E36 and E49 fetuses. Scale bars, 3.7 mm. NA, non-examination. ( E ) Agarose gel electrophoresis of the PCR products from all knockout monkey samples. BRN2 +/+ sample (123044-1#) is shown as a negative control of the target gene editing. P, placenta; B, brain cells. ( F ) Immunostainings of BRN2 and SOX1 in the BRN2 +/+ and BRN2 −/− monkey fetal cortex, confirming BRN2 deletion in the BRN2 −/− cortex. Blue, DAPI, nuclear staining. Scale bars, 100 μm. IHC, immunohistochemistry.

    Techniques Used: Knock-Out, Agarose Gel Electrophoresis, Negative Control, Staining, Immunohistochemistry

    ( A to D ) The coronal sections from the same region of primary somatosensory cortices were used to perform comparison analysis between E49 BRN2 +/+ and BRN2 −/− telencephalon (see Materials and Methods). (A) SOX2 and PAX6 expressions in the BRN2 −/− and BRN2 +/+ cortex, respectively. (B and B′) Representative staining images (B) and quantification (B′) of PVI, KI67, and DAPI in BRN2 −/− and BRN2 +/+ cortical cells. DAPI staining was used to identify cell division orientation. Quantification data of division orientations of RGCs are presented as means ± SEM ( n = 6 representative images; >100 cells per group were counted, * P < 0.01). (C) Representative staining images of TUJ1 and SOX1 in BRN2 −/− and BRN2 +/+ cortical cells, respectively. (D and D′) HOPX and SOX1 expressions in the BRN2 −/− and BRN2 +/+ cortex, respectively. Right images are higher magnifications of left images. (D′) Quantification of HOPX cell migration into the SVZ region from VZ region (means ± SEM, n = 4 representative images; >500 cells per group were counted, * P < 0.01). Scale bars, 100 μm. ( E and F ) Visualization of major classes of neural cells from BRN2 +/+ and BRN2 −/− single cells across three different stages by Uniform Manifold Approximation and Projection (UMAP) analysis. ( G ) Plots of classical markers representing RGC ( SOX2 ), intermediate progenitor (IP) ( EOMES ), and excitatory neuron (ExN) ( STMN2 ), respectively. ( H ) Proportion dynamic of each cell type over cortical development. ( I ) Developmental trajectory of cortical cells across three different stages. RG-IP-N (RGC-IP-to-neuron) is the indirect way, while RG-N (RGC-to-neuron) is the direct way. The dotted lines show developmental trajectories of RGCs. The arrowheads show the directions of these trajectories. ( J ) DEGs between the BRN2 −/− and BRN2 +/+ cortex (see table S3). Representative transcription factors (TFs), gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways are shown. Blue, DAPI, nuclear staining.
    Figure Legend Snippet: ( A to D ) The coronal sections from the same region of primary somatosensory cortices were used to perform comparison analysis between E49 BRN2 +/+ and BRN2 −/− telencephalon (see Materials and Methods). (A) SOX2 and PAX6 expressions in the BRN2 −/− and BRN2 +/+ cortex, respectively. (B and B′) Representative staining images (B) and quantification (B′) of PVI, KI67, and DAPI in BRN2 −/− and BRN2 +/+ cortical cells. DAPI staining was used to identify cell division orientation. Quantification data of division orientations of RGCs are presented as means ± SEM ( n = 6 representative images; >100 cells per group were counted, * P < 0.01). (C) Representative staining images of TUJ1 and SOX1 in BRN2 −/− and BRN2 +/+ cortical cells, respectively. (D and D′) HOPX and SOX1 expressions in the BRN2 −/− and BRN2 +/+ cortex, respectively. Right images are higher magnifications of left images. (D′) Quantification of HOPX cell migration into the SVZ region from VZ region (means ± SEM, n = 4 representative images; >500 cells per group were counted, * P < 0.01). Scale bars, 100 μm. ( E and F ) Visualization of major classes of neural cells from BRN2 +/+ and BRN2 −/− single cells across three different stages by Uniform Manifold Approximation and Projection (UMAP) analysis. ( G ) Plots of classical markers representing RGC ( SOX2 ), intermediate progenitor (IP) ( EOMES ), and excitatory neuron (ExN) ( STMN2 ), respectively. ( H ) Proportion dynamic of each cell type over cortical development. ( I ) Developmental trajectory of cortical cells across three different stages. RG-IP-N (RGC-IP-to-neuron) is the indirect way, while RG-N (RGC-to-neuron) is the direct way. The dotted lines show developmental trajectories of RGCs. The arrowheads show the directions of these trajectories. ( J ) DEGs between the BRN2 −/− and BRN2 +/+ cortex (see table S3). Representative transcription factors (TFs), gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways are shown. Blue, DAPI, nuclear staining.

    Techniques Used: Staining, Migration

    ( A ) BRN2 and COUP-TFII staining showed the wide distribution of BRN2 in the telencephalic ganglionic eminences (GEs). PSB, pallial-subpallial boundary. ( B ) NKX2-1 and COUP-TFII expression in BRN2 +/+ and BRN2 −/− GEs, showing that COUP-TFII was specifically expressed in BRN2 +/+ CGE/LGE, whereas it was obviously activated in the whole BRN2 −/− GEs (including both LGE/CGE and MGE). The sections of BRN2 +/+ and BRN2 −/− were from equivalent coronal levels (details are in Materials and Methods). ( C ) Visualization of major classes of interneuron and interneuron progenitor (iNP) by UMAP analysis. ( D ) Developmental trajectory of interneurons and iNPs showing that BRN2 loss changed interneuron development trajectory. Cells in cluster 3 were markedly decreased in BRN2 −/− monkeys. ( E ) DEGs between BRN2 +/+ and BRN2 −/− interneurons and iNPs (table S4). Representative transcription factors (TFs), GO terms, and KEGG pathways are shown. ( F and G ) The sections of BRN2 +/+ and BRN2 −/− telencephalon were from equivalent coronal levels. (F and G) Representative staining images of HOPX and SOX1 in BRN2 +/+ (F) and BRN2 −/− (G) GEs. F1 and G1 are magnifications of the squares in (F) and (G). ( H ) PAX6 and SOX2 expression in BRN2 +/+ and BRN2 −/− GEs. ( I ) SST and NKX2-1 expression in BRN2 +/+ and BRN2 −/− GEs, respectively. ( J ) CALRETININ (CR) expression in BRN2 +/+ and BRN2 −/− telencephalon. Arrows indicate the migration orientations of CR interneurons. All images of immunofluorescence were from coronal sections spanning the rostral-caudal extent of the telencephalon. Scale bars, 500 μm. Blue, DAPI, nuclear staining.
    Figure Legend Snippet: ( A ) BRN2 and COUP-TFII staining showed the wide distribution of BRN2 in the telencephalic ganglionic eminences (GEs). PSB, pallial-subpallial boundary. ( B ) NKX2-1 and COUP-TFII expression in BRN2 +/+ and BRN2 −/− GEs, showing that COUP-TFII was specifically expressed in BRN2 +/+ CGE/LGE, whereas it was obviously activated in the whole BRN2 −/− GEs (including both LGE/CGE and MGE). The sections of BRN2 +/+ and BRN2 −/− were from equivalent coronal levels (details are in Materials and Methods). ( C ) Visualization of major classes of interneuron and interneuron progenitor (iNP) by UMAP analysis. ( D ) Developmental trajectory of interneurons and iNPs showing that BRN2 loss changed interneuron development trajectory. Cells in cluster 3 were markedly decreased in BRN2 −/− monkeys. ( E ) DEGs between BRN2 +/+ and BRN2 −/− interneurons and iNPs (table S4). Representative transcription factors (TFs), GO terms, and KEGG pathways are shown. ( F and G ) The sections of BRN2 +/+ and BRN2 −/− telencephalon were from equivalent coronal levels. (F and G) Representative staining images of HOPX and SOX1 in BRN2 +/+ (F) and BRN2 −/− (G) GEs. F1 and G1 are magnifications of the squares in (F) and (G). ( H ) PAX6 and SOX2 expression in BRN2 +/+ and BRN2 −/− GEs. ( I ) SST and NKX2-1 expression in BRN2 +/+ and BRN2 −/− GEs, respectively. ( J ) CALRETININ (CR) expression in BRN2 +/+ and BRN2 −/− telencephalon. Arrows indicate the migration orientations of CR interneurons. All images of immunofluorescence were from coronal sections spanning the rostral-caudal extent of the telencephalon. Scale bars, 500 μm. Blue, DAPI, nuclear staining.

    Techniques Used: Staining, Expressing, Migration, Immunofluorescence

    ( A ) Schematic of generating BRN2 -knockout ( BRN2 −/− ) human embryonic stem cells (ESCs) by CRISPR-Cas9 gene editing. ( B ) Generation of cortical organoids using human wild-type ( BRN2 +/+ ) and BRN2 −/− ESCs, respectively, showing that BRN2 −/− ESCs lost the ability to generate cortical organoids. The top panel shows the diagram of cortical organoid generation. ( C ) Expression of representative neuroepithelium markers in cortical organoids from BRN2 +/+ and BRN2 −/− ESCs. ( D ) Comparison of proliferation markers KI67 and PVI in BRN2 +/+ or BRN2 −/− cortical organoids. DAPI staining was used to identify cell division orientation. ( E ) Quantifications of cell division orientation in BRN2 +/+ and BRN2 −/− cells of cortical organoids (means ± SEM, n = 4 independent experiments; >200 cells per group were counted), respectively. ( F ) Comparison of CUX1 (an upper-layer neuron marker) and FOXP2 (a deep-layer neuron marker) expressions between BRN2 +/+ and BRN2 −/− cortical organoids, respectively. ( G ) Quantification of SOX2-, PAX6-, CUX1-, and FOXP2-positive cells in BRN2 +/+ and BRN2 −/− cortical organoids, respectively. Data are presented as means ± SEM, n = 3 slices; >300 cells per group were counted. * P < 0.05. Scale bars, 100 μm.
    Figure Legend Snippet: ( A ) Schematic of generating BRN2 -knockout ( BRN2 −/− ) human embryonic stem cells (ESCs) by CRISPR-Cas9 gene editing. ( B ) Generation of cortical organoids using human wild-type ( BRN2 +/+ ) and BRN2 −/− ESCs, respectively, showing that BRN2 −/− ESCs lost the ability to generate cortical organoids. The top panel shows the diagram of cortical organoid generation. ( C ) Expression of representative neuroepithelium markers in cortical organoids from BRN2 +/+ and BRN2 −/− ESCs. ( D ) Comparison of proliferation markers KI67 and PVI in BRN2 +/+ or BRN2 −/− cortical organoids. DAPI staining was used to identify cell division orientation. ( E ) Quantifications of cell division orientation in BRN2 +/+ and BRN2 −/− cells of cortical organoids (means ± SEM, n = 4 independent experiments; >200 cells per group were counted), respectively. ( F ) Comparison of CUX1 (an upper-layer neuron marker) and FOXP2 (a deep-layer neuron marker) expressions between BRN2 +/+ and BRN2 −/− cortical organoids, respectively. ( G ) Quantification of SOX2-, PAX6-, CUX1-, and FOXP2-positive cells in BRN2 +/+ and BRN2 −/− cortical organoids, respectively. Data are presented as means ± SEM, n = 3 slices; >300 cells per group were counted. * P < 0.05. Scale bars, 100 μm.

    Techniques Used: Knock-Out, CRISPR, Expressing, Staining, Marker

    ( A and B ) Comparison of BRN1 expressions between BRN2 +/+ and BRN2 −/− human ESC–derived cortical organoids (A) and between BRN2 +/+ and BRN2 −/− monkey cortical cells (B). Data are presented as means ± SEM ( n = 3). NS, no significance, P > 0.05. ( C ) Gene plots show densities of BRN2, polymerase II (Pol II), and control (IgG) at the SOX2 promoter in human developing cortex, indicating that BRN2 binds to the promoter region of SOX2 . ( D ) Gene plots show densities of BRN2, Pol II, and control (IgG) at the STAT3 promoter in human developing cortex, indicating that BRN2 regulates STAT3 expression by directly binding to the promoter region of STAT3 . ( E ) Comparison of STAT3 expressions in the BRN2 +/+ and BRN2 −/− cortex, which exhibited a significant down-regulation in the BRN2 −/− cortex. ** P < 0.01. ( F ) Summary of BRN2 regulatory mechanisms on monkey telencephalon development. aRGC, apical RGC. The thickness and fineness of the line represent the strong and weak activity, respectively. Arrows indicate the activation, and trident lines indicate the repression.
    Figure Legend Snippet: ( A and B ) Comparison of BRN1 expressions between BRN2 +/+ and BRN2 −/− human ESC–derived cortical organoids (A) and between BRN2 +/+ and BRN2 −/− monkey cortical cells (B). Data are presented as means ± SEM ( n = 3). NS, no significance, P > 0.05. ( C ) Gene plots show densities of BRN2, polymerase II (Pol II), and control (IgG) at the SOX2 promoter in human developing cortex, indicating that BRN2 binds to the promoter region of SOX2 . ( D ) Gene plots show densities of BRN2, Pol II, and control (IgG) at the STAT3 promoter in human developing cortex, indicating that BRN2 regulates STAT3 expression by directly binding to the promoter region of STAT3 . ( E ) Comparison of STAT3 expressions in the BRN2 +/+ and BRN2 −/− cortex, which exhibited a significant down-regulation in the BRN2 −/− cortex. ** P < 0.01. ( F ) Summary of BRN2 regulatory mechanisms on monkey telencephalon development. aRGC, apical RGC. The thickness and fineness of the line represent the strong and weak activity, respectively. Arrows indicate the activation, and trident lines indicate the repression.

    Techniques Used: Derivative Assay, Expressing, Binding Assay, Activity Assay, Activation Assay

    anti brn2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti brn2
    MUC1 associates with MYC and decreased survival in the TCGA-PAAD dataset. (A) GSEA of the 163 PDAC tumors in the TCGA-PAAD/PDAC dataset for associations of MUC1 expression with the indicated HALLMARK gene signatures. (B–D) Enrichment plots for the HALLMARK INTERFERON ALPHA RESPONSE ( B ) HALLMARK MYC TARGETS V1 ( C ) and HALLMARK NOTCH SIGNALING ( D ) pathways, comparing MUC1-high to MUC1-low PDAC tumors in the TCGA-PAAD/PDAC dataset. ( E ) Probability of survival comparing MUC1-high, MYC-high (blue curve) to MUC1-low, MYC-high (red curve) PDAC tumors in the TCGA-PAAD/PDAC dataset. ( F ) Probability of survival comparing MUC1-high, MYC-low (blue curve) to MUC1-low, MYC-low (red curve) PDAC tumors in the TCGA-PAAD/PDAC dataset. ( G ) Schematic representation of MUC1-C in driving PDAC NE lineage specification. MUC1-C induces STAT1 and the type I and II IFN pathways, which are associated with KRAS mutant PDAC tumors and poor patient outcomes . MUC1-C also activates MYC, which is required for oncogenic mutant KRAS signaling in PDACs. Further studies are needed, at least in part, to determine whether MUC1-C independently activates STAT1 and MYC. The present data further indicate that MUC1-C integrates MYC activation with induction of the (i) OCT4, SOX2 and KLF4 pluripotency factors, (ii) <t>BRN2,</t> ASCL1 and AURKA NE markers, and (iii) NOTCH1/2 stemness TFs. In this way, MUC1-C promotes NE dedifferentiation, self-renewal capacity and tumorigenicity in PDAC progression.
    Anti Brn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "MUC1-C dictates neuroendocrine lineage specification in pancreatic ductal adenocarcinomas"

    Article Title: MUC1-C dictates neuroendocrine lineage specification in pancreatic ductal adenocarcinomas

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgab097

    MUC1 associates with MYC and decreased survival in the TCGA-PAAD dataset. (A) GSEA of the 163 PDAC tumors in the TCGA-PAAD/PDAC dataset for associations of MUC1 expression with the indicated HALLMARK gene signatures. (B–D) Enrichment plots for the HALLMARK INTERFERON ALPHA RESPONSE ( B ) HALLMARK MYC TARGETS V1 ( C ) and HALLMARK NOTCH SIGNALING ( D ) pathways, comparing MUC1-high to MUC1-low PDAC tumors in the TCGA-PAAD/PDAC dataset. ( E ) Probability of survival comparing MUC1-high, MYC-high (blue curve) to MUC1-low, MYC-high (red curve) PDAC tumors in the TCGA-PAAD/PDAC dataset. ( F ) Probability of survival comparing MUC1-high, MYC-low (blue curve) to MUC1-low, MYC-low (red curve) PDAC tumors in the TCGA-PAAD/PDAC dataset. ( G ) Schematic representation of MUC1-C in driving PDAC NE lineage specification. MUC1-C induces STAT1 and the type I and II IFN pathways, which are associated with KRAS mutant PDAC tumors and poor patient outcomes . MUC1-C also activates MYC, which is required for oncogenic mutant KRAS signaling in PDACs. Further studies are needed, at least in part, to determine whether MUC1-C independently activates STAT1 and MYC. The present data further indicate that MUC1-C integrates MYC activation with induction of the (i) OCT4, SOX2 and KLF4 pluripotency factors, (ii) BRN2, ASCL1 and AURKA NE markers, and (iii) NOTCH1/2 stemness TFs. In this way, MUC1-C promotes NE dedifferentiation, self-renewal capacity and tumorigenicity in PDAC progression.
    Figure Legend Snippet: MUC1 associates with MYC and decreased survival in the TCGA-PAAD dataset. (A) GSEA of the 163 PDAC tumors in the TCGA-PAAD/PDAC dataset for associations of MUC1 expression with the indicated HALLMARK gene signatures. (B–D) Enrichment plots for the HALLMARK INTERFERON ALPHA RESPONSE ( B ) HALLMARK MYC TARGETS V1 ( C ) and HALLMARK NOTCH SIGNALING ( D ) pathways, comparing MUC1-high to MUC1-low PDAC tumors in the TCGA-PAAD/PDAC dataset. ( E ) Probability of survival comparing MUC1-high, MYC-high (blue curve) to MUC1-low, MYC-high (red curve) PDAC tumors in the TCGA-PAAD/PDAC dataset. ( F ) Probability of survival comparing MUC1-high, MYC-low (blue curve) to MUC1-low, MYC-low (red curve) PDAC tumors in the TCGA-PAAD/PDAC dataset. ( G ) Schematic representation of MUC1-C in driving PDAC NE lineage specification. MUC1-C induces STAT1 and the type I and II IFN pathways, which are associated with KRAS mutant PDAC tumors and poor patient outcomes . MUC1-C also activates MYC, which is required for oncogenic mutant KRAS signaling in PDACs. Further studies are needed, at least in part, to determine whether MUC1-C independently activates STAT1 and MYC. The present data further indicate that MUC1-C integrates MYC activation with induction of the (i) OCT4, SOX2 and KLF4 pluripotency factors, (ii) BRN2, ASCL1 and AURKA NE markers, and (iii) NOTCH1/2 stemness TFs. In this way, MUC1-C promotes NE dedifferentiation, self-renewal capacity and tumorigenicity in PDAC progression.

    Techniques Used: Expressing, Mutagenesis, Activation Assay

    brn2 d2c1l rabbit mab 12137  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc brn2 d2c1l rabbit mab 12137
    A Nuclear localization of H19 in NCI-H660. y -Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, <t>BRN2,</t> and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16D CRPC and AdPC cell line LNCaP after transient overexpression of H19 . The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16D CRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19 FL (full-length H19 ) or H19 DEL (5′ deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD ( A , C ), or mean ± SEM ( E ); n = 3 ( A , C , E ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.
    Brn2 D2c1l Rabbit Mab 12137, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer"

    Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

    Journal: Nature Communications

    doi: 10.1038/s41467-021-26901-9

    A Nuclear localization of H19 in NCI-H660. y -Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, BRN2, and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16D CRPC and AdPC cell line LNCaP after transient overexpression of H19 . The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16D CRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19 FL (full-length H19 ) or H19 DEL (5′ deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD ( A , C ), or mean ± SEM ( E ); n = 3 ( A , C , E ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.
    Figure Legend Snippet: A Nuclear localization of H19 in NCI-H660. y -Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, BRN2, and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16D CRPC and AdPC cell line LNCaP after transient overexpression of H19 . The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16D CRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19 FL (full-length H19 ) or H19 DEL (5′ deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD ( A , C ), or mean ± SEM ( E ); n = 3 ( A , C , E ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.

    Techniques Used: Over Expression, Binding Assay, Stable Transfection, Expressing, Two Tailed Test

    A Assessment of sensitivity and specificity of H19 in NEPC for use as a diagnostic along with other recently identified NEPC oncogenes (BRN2, SRRM4, and PEG10). Arrows denote patients that are negative for CHGA (black) or CHGA, SYP, and NSE (red) yet positive for H19 . B , C , Kaplan–Meir estimates for the impact of H19 on prostate cancer clinical outcomes. B Survival analysis for H19 in ADT untreated patients and C ADT treated patients for biochemical recurrence (BCR—left panels) and metastasis (MET—right panels). Samples were split by H19 tertile expression to test if either of these subgroups of patients were more likely to develop BCR and MET in the context of treatment. P values were calculated using a Kaplan–Meier estimator statistical test to determine significance for the observed stratification in probabilities across the three subgroups in each panel. D , Illustration of H19 transcription and mechanism of action during PCa, NEtD, and tNEPC. During early stage disease, adenocarcinoma cancer cells are driven by an active AR bound to androgens that prevent the transcription of H19 . As the disease progresses (gray triangle), ADTs suppress AR activity, SOX2 increases, and results in the persistent transcription, expression, and activation of H19 . Once active, H19 operates via dual epigenetic mechanisms; (on the left) binds PRC2 complex members, aiding in altering methylation on H3K4Me3/H3K27Me3 histone marks of PRC2 target genes and (on the right) genome-wide alteration of methylation at CpG sites on DNA. Collectively, these epigenetic changes result in the activation of NE gene expression and deactivation of AR signaling gene expression.
    Figure Legend Snippet: A Assessment of sensitivity and specificity of H19 in NEPC for use as a diagnostic along with other recently identified NEPC oncogenes (BRN2, SRRM4, and PEG10). Arrows denote patients that are negative for CHGA (black) or CHGA, SYP, and NSE (red) yet positive for H19 . B , C , Kaplan–Meir estimates for the impact of H19 on prostate cancer clinical outcomes. B Survival analysis for H19 in ADT untreated patients and C ADT treated patients for biochemical recurrence (BCR—left panels) and metastasis (MET—right panels). Samples were split by H19 tertile expression to test if either of these subgroups of patients were more likely to develop BCR and MET in the context of treatment. P values were calculated using a Kaplan–Meier estimator statistical test to determine significance for the observed stratification in probabilities across the three subgroups in each panel. D , Illustration of H19 transcription and mechanism of action during PCa, NEtD, and tNEPC. During early stage disease, adenocarcinoma cancer cells are driven by an active AR bound to androgens that prevent the transcription of H19 . As the disease progresses (gray triangle), ADTs suppress AR activity, SOX2 increases, and results in the persistent transcription, expression, and activation of H19 . Once active, H19 operates via dual epigenetic mechanisms; (on the left) binds PRC2 complex members, aiding in altering methylation on H3K4Me3/H3K27Me3 histone marks of PRC2 target genes and (on the right) genome-wide alteration of methylation at CpG sites on DNA. Collectively, these epigenetic changes result in the activation of NE gene expression and deactivation of AR signaling gene expression.

    Techniques Used: Diagnostic Assay, Expressing, Activity Assay, Activation Assay, Methylation, Genome Wide

    rabbit anti brn2 pou3f2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti brn2 pou3f2
    Loss of podoplanin restores pigmentation and melanocyte differentiation (A and B) Tumors (A) and cells pellets (B) of PDPN + and PDPN KO B16F10 cell lines. (C) Imaging of pigmentation (brightfield; left) of PDPN + and PDPN KO B16F10 cell lines, labeled with mOrange (red) or CFP (blue) respectively (middle). The scale bar represents 50 microns. (D) Heatmaps showing expression ( Z score) of podoplanin ( PDPN ) and eight dedifferentiation-associated genes in datasets of primary tumor samples of melanoma patients (top; Riker et al. GSE7553 ) and metastatic melanoma cultures (bottom; Mannheim cohort, GSE4843 ; from <xref ref-type=Hoek et al., 2006 ). For the Mannheim cohort, each number indicates a separate metastatic melanoma culture, and the BRAF and/or NRAS mutation status is indicated for each. (E) mRNA expression of five melanocyte-associated ( Mlana/Mart-1 (p = 0.2045), Tyr (p = 0.9276), Dct (p = 0.9859), Gpnmb (p = 0.1308), Pmel (p = 0.9994); left) and three invasion-associated ( Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in PDPN + B16F10 cells. mRNA expression is calculated as fold change of Gapdh expression and normalized to expression in PDPN KO B16F10 cells (set at 1 as indicated by dashed line). Data shown as mean with dots representing n = 3 biological replicates. Two-way ANOVA with Sidak's multiple comparisons; p values indicated above. (F) Western blot analysis of Podoplanin, Melan-A, Tyrosinase and Brn2 (encoded by Pou3f2 gene) in PDPN KO and PDPN + B16F10 cells. Tubulin and histone H3 are used as loading controls. " width="250" height="auto" />
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    Images

    1) Product Images from "Podoplanin drives dedifferentiation and amoeboid invasion of melanoma"

    Article Title: Podoplanin drives dedifferentiation and amoeboid invasion of melanoma

    Journal: iScience

    doi: 10.1016/j.isci.2021.102976

    Loss of podoplanin restores pigmentation and melanocyte differentiation (A and B) Tumors (A) and cells pellets (B) of PDPN + and PDPN KO B16F10 cell lines. (C) Imaging of pigmentation (brightfield; left) of PDPN + and PDPN KO B16F10 cell lines, labeled with mOrange (red) or CFP (blue) respectively (middle). The scale bar represents 50 microns. (D) Heatmaps showing expression ( Z score) of podoplanin ( PDPN ) and eight dedifferentiation-associated genes in datasets of primary tumor samples of melanoma patients (top; Riker et al. GSE7553 ) and metastatic melanoma cultures (bottom; Mannheim cohort, GSE4843 ; from <xref ref-type=Hoek et al., 2006 ). For the Mannheim cohort, each number indicates a separate metastatic melanoma culture, and the BRAF and/or NRAS mutation status is indicated for each. (E) mRNA expression of five melanocyte-associated ( Mlana/Mart-1 (p = 0.2045), Tyr (p = 0.9276), Dct (p = 0.9859), Gpnmb (p = 0.1308), Pmel (p = 0.9994); left) and three invasion-associated ( Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in PDPN + B16F10 cells. mRNA expression is calculated as fold change of Gapdh expression and normalized to expression in PDPN KO B16F10 cells (set at 1 as indicated by dashed line). Data shown as mean with dots representing n = 3 biological replicates. Two-way ANOVA with Sidak's multiple comparisons; p values indicated above. (F) Western blot analysis of Podoplanin, Melan-A, Tyrosinase and Brn2 (encoded by Pou3f2 gene) in PDPN KO and PDPN + B16F10 cells. Tubulin and histone H3 are used as loading controls. " title="... Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Loss of podoplanin restores pigmentation and melanocyte differentiation (A and B) Tumors (A) and cells pellets (B) of PDPN + and PDPN KO B16F10 cell lines. (C) Imaging of pigmentation (brightfield; left) of PDPN + and PDPN KO B16F10 cell lines, labeled with mOrange (red) or CFP (blue) respectively (middle). The scale bar represents 50 microns. (D) Heatmaps showing expression ( Z score) of podoplanin ( PDPN ) and eight dedifferentiation-associated genes in datasets of primary tumor samples of melanoma patients (top; Riker et al. GSE7553 ) and metastatic melanoma cultures (bottom; Mannheim cohort, GSE4843 ; from Hoek et al., 2006 ). For the Mannheim cohort, each number indicates a separate metastatic melanoma culture, and the BRAF and/or NRAS mutation status is indicated for each. (E) mRNA expression of five melanocyte-associated ( Mlana/Mart-1 (p = 0.2045), Tyr (p = 0.9276), Dct (p = 0.9859), Gpnmb (p = 0.1308), Pmel (p = 0.9994); left) and three invasion-associated ( Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in PDPN + B16F10 cells. mRNA expression is calculated as fold change of Gapdh expression and normalized to expression in PDPN KO B16F10 cells (set at 1 as indicated by dashed line). Data shown as mean with dots representing n = 3 biological replicates. Two-way ANOVA with Sidak's multiple comparisons; p values indicated above. (F) Western blot analysis of Podoplanin, Melan-A, Tyrosinase and Brn2 (encoded by Pou3f2 gene) in PDPN KO and PDPN + B16F10 cells. Tubulin and histone H3 are used as loading controls.

    Techniques Used: Imaging, Labeling, Expressing, Mutagenesis, Western Blot

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    Cell Signaling Technology Inc brn2
    ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and <t>BRN2.</t> MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.
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    Differentiation of SMNs, cortical neurons, and sensory neurons using inducible transcription factors. (A) Distinct tet-inducible transcription factors contained within PiggyBac constructs were nucleofected into iPSCs to generate different cell types. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1 staining. Darker, lighter, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C) Cyclic immunofluorescence of SMNs for HB9, NEUN, ISL1, TUJ1, and two different CHAT antibodies; quantified in (D). “Combined” indicates the percentage of cells positive for all markers. (E) Cortical neurons stained for <t>BRN2,</t> FOXG1, and TUJ1; quantified (F). (G) Sensory neurons stained for BRN3A, ISL1, and TUJ1; quantified in (H). Scale bars = 100uM; n=8 wells for all groups. Across panels, arrows indicate the same neuron, which is magnified in the inset.
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    Differentiation of SMNs, cortical neurons, and sensory neurons using inducible transcription factors. (A) Distinct tet-inducible transcription factors contained within PiggyBac constructs were nucleofected into iPSCs to generate different cell types. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1 staining. Darker, lighter, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C) Cyclic immunofluorescence of SMNs for HB9, NEUN, ISL1, TUJ1, and two different CHAT antibodies; quantified in (D). “Combined” indicates the percentage of cells positive for all markers. (E) Cortical neurons stained for <t>BRN2,</t> FOXG1, and TUJ1; quantified (F). (G) Sensory neurons stained for BRN3A, ISL1, and TUJ1; quantified in (H). Scale bars = 100uM; n=8 wells for all groups. Across panels, arrows indicate the same neuron, which is magnified in the inset.
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    NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C) . (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker <t>BRN2</t> (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G i ) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G ii ) . (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10 8 ) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I) , single stained with DiI (J) , and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.
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    NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C) . (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker <t>BRN2</t> (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G i ) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G ii ) . (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10 8 ) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I) , single stained with DiI (J) , and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.
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    A Nuclear localization of H19 in NCI-H660. y -Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, <t>BRN2,</t> and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16D CRPC and AdPC cell line LNCaP after transient overexpression of H19 . The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16D CRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19 FL (full-length H19 ) or H19 DEL (5′ deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD ( A , C ), or mean ± SEM ( E ); n = 3 ( A , C , E ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.
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    Loss of podoplanin restores pigmentation and melanocyte differentiation (A and B) Tumors (A) and cells pellets (B) of PDPN + and PDPN KO B16F10 cell lines. (C) Imaging of pigmentation (brightfield; left) of PDPN + and PDPN KO B16F10 cell lines, labeled with mOrange (red) or CFP (blue) respectively (middle). The scale bar represents 50 microns. (D) Heatmaps showing expression ( Z score) of podoplanin ( PDPN ) and eight dedifferentiation-associated genes in datasets of primary tumor samples of melanoma patients (top; Riker et al. GSE7553 ) and metastatic melanoma cultures (bottom; Mannheim cohort, GSE4843 ; from <xref ref-type=Hoek et al., 2006 ). For the Mannheim cohort, each number indicates a separate metastatic melanoma culture, and the BRAF and/or NRAS mutation status is indicated for each. (E) mRNA expression of five melanocyte-associated ( Mlana/Mart-1 (p = 0.2045), Tyr (p = 0.9276), Dct (p = 0.9859), Gpnmb (p = 0.1308), Pmel (p = 0.9994); left) and three invasion-associated ( Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in PDPN + B16F10 cells. mRNA expression is calculated as fold change of Gapdh expression and normalized to expression in PDPN KO B16F10 cells (set at 1 as indicated by dashed line). Data shown as mean with dots representing n = 3 biological replicates. Two-way ANOVA with Sidak's multiple comparisons; p values indicated above. (F) Western blot analysis of Podoplanin, Melan-A, Tyrosinase and Brn2 (encoded by Pou3f2 gene) in PDPN KO and PDPN + B16F10 cells. Tubulin and histone H3 are used as loading controls. " width="250" height="auto" />
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    Image Search Results


    ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and BRN2. MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.

    Journal: bioRxiv

    Article Title: Deletion of TRPC6 , an autism risk gene, induces hyperexcitability in cortical neurons derived from human pluripotent stem cells

    doi: 10.1101/2022.11.14.516407

    Figure Lengend Snippet: ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and BRN2. MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.

    Article Snippet: Primary antibodies consisted of SOX2 (Rabbit, 1:200, Invitrogen: MA1-014), FOXG1 (Rabbit, 1:200, Abcam: ab18259), OTX2 (Goat, 1:300, R&D: AF1979), Nestin (Mouse, 1:100, Invitrogen: MA1110, MAP2 (chicken, 1:500, Abcam: ab5392), MAP2 (Mouse, 1:500, Invitrogen: 13-1500), beta tubulin III (Mouse, 1:300, MAB1637), TBR2 (Rabbit,1:300, Cell signaling: 66325), CTIP2 (Rabbit, 1:200, Cell signaling:), BRN2 (Rabbit, 1:200, Cell signaling:12137), GFAP (Chicken, 1:400, Abcam: ab4674), and SATB2 (Rabbit, 1:200, Invitrogen:PA5-83092).

    Techniques: Immunostaining, Clone Assay, Marker, Staining, Expressing

    Differentiation of SMNs, cortical neurons, and sensory neurons using inducible transcription factors. (A) Distinct tet-inducible transcription factors contained within PiggyBac constructs were nucleofected into iPSCs to generate different cell types. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1 staining. Darker, lighter, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C) Cyclic immunofluorescence of SMNs for HB9, NEUN, ISL1, TUJ1, and two different CHAT antibodies; quantified in (D). “Combined” indicates the percentage of cells positive for all markers. (E) Cortical neurons stained for BRN2, FOXG1, and TUJ1; quantified (F). (G) Sensory neurons stained for BRN3A, ISL1, and TUJ1; quantified in (H). Scale bars = 100uM; n=8 wells for all groups. Across panels, arrows indicate the same neuron, which is magnified in the inset.

    Journal: bioRxiv

    Article Title: iPSC Motor Neurons with Familial ALS Mutations Capture Gene Expression Changes in Postmortem Sporadic ALS Motor Neurons

    doi: 10.1101/2022.10.25.513780

    Figure Lengend Snippet: Differentiation of SMNs, cortical neurons, and sensory neurons using inducible transcription factors. (A) Distinct tet-inducible transcription factors contained within PiggyBac constructs were nucleofected into iPSCs to generate different cell types. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1 staining. Darker, lighter, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C) Cyclic immunofluorescence of SMNs for HB9, NEUN, ISL1, TUJ1, and two different CHAT antibodies; quantified in (D). “Combined” indicates the percentage of cells positive for all markers. (E) Cortical neurons stained for BRN2, FOXG1, and TUJ1; quantified (F). (G) Sensory neurons stained for BRN3A, ISL1, and TUJ1; quantified in (H). Scale bars = 100uM; n=8 wells for all groups. Across panels, arrows indicate the same neuron, which is magnified in the inset.

    Article Snippet: The primary antibodies used in this study were: anti-Brn2 (1:1000, Cell Signaling 12137S), anti-FoxG1 (1:500, Millipore Sigma MABD79), anti-Tuj1 (1:500, Aves TUJ-0020), anti-Brn3a (1:200, Millipore MAB1585), anti-Isl1 (1:200, Abcam ab203406), anti-GFAP (1:250, abcam ab194324), anti-S100β (1:200, abcam ab52642), and anti-TDP-43 (1:500, proteintech 10782-2-AP).

    Techniques: Construct, Staining, Immunofluorescence

    NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C) . (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker BRN2 (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G i ) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G ii ) . (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10 8 ) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I) , single stained with DiI (J) , and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.

    Journal: Frontiers in Integrative Neuroscience

    Article Title: Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers

    doi: 10.3389/fnint.2022.879832

    Figure Lengend Snippet: NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C) . (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker BRN2 (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G i ) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G ii ) . (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10 8 ) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I) , single stained with DiI (J) , and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.

    Article Snippet: For immunostaining, the freezing medium was washed with PBS, and tissues were permeabilized with 0.3% Triton-X in PBS for 40 min. Tissues were blocked in 3% BSA in PBS containing 0.2% Tween-20 (PBST) for 2 h. Primary antibodies BRN2 (1:200, cell signaling, 12137), CTIP2 (1:200, cell signaling, 12120), and SOX2 (1:300, Invitrogen, MA1-014) were diluted in blocking solution and added to the tissue overnight at 4°C.

    Techniques: Marker, Flow Cytometry, Staining, Labeling, Derivative Assay, Cell Culture, Immunostaining, Microscopy, Western Blot, Isolation

    A Nuclear localization of H19 in NCI-H660. y -Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, BRN2, and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16D CRPC and AdPC cell line LNCaP after transient overexpression of H19 . The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16D CRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19 FL (full-length H19 ) or H19 DEL (5′ deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD ( A , C ), or mean ± SEM ( E ); n = 3 ( A , C , E ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.

    Journal: Nature Communications

    Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

    doi: 10.1038/s41467-021-26901-9

    Figure Lengend Snippet: A Nuclear localization of H19 in NCI-H660. y -Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, BRN2, and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16D CRPC and AdPC cell line LNCaP after transient overexpression of H19 . The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16D CRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19 FL (full-length H19 ) or H19 DEL (5′ deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD ( A , C ), or mean ± SEM ( E ); n = 3 ( A , C , E ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.

    Article Snippet: Antibodies used: SOX2 (sc-365823, Santa Cruz Biotechnology, 1:400 dilution), H3K27me3 (9733S, Cell Signaling Technology, 1:1000 dilution), EZH2 ((D2C9) XP Rabbit mAb (5246, Cell Signaling Technology, 1:1000 dilution), NSE (sc-21738, Santa Cruz Biotechnology, 1:500 dilution), Synaptophysin (sc-365488, Santa Cruz Biotechnology, 1:500 dilution), CHGA (60893S, Cell Signaling Technology, 1:1000 dilution), BRN2 ((D2C1L) Rabbit mAb 12137, Cell Signaling Technology, 1:1000 dilution), H3 ((D1H2) XP ® Rabbit mAb 4499, Cell Signaling Technology, 1:1000 dilution), Androgen receptor (5153S, Cell Signaling Technology, 1:1000 dilution), H3K4me3 (ab8580, abcam, 1:1000 dilution), P53 (2527 S, Cell Signaling Technology, 1:1000 dilution), Rb (9313S, Cell Signaling Technology, 1:1000 dilution), CK8 (sc–57004, Santa Cruz Biotechnology, 1:300 dilution), PSA (sc-7316, Santa Cruz Biotechnology, 1:250 dilution), HRP conjugated anti-β-actin (Cat. no. A3854, Sigma, 1:10,000 dilution).

    Techniques: Over Expression, Binding Assay, Stable Transfection, Expressing, Two Tailed Test

    A Assessment of sensitivity and specificity of H19 in NEPC for use as a diagnostic along with other recently identified NEPC oncogenes (BRN2, SRRM4, and PEG10). Arrows denote patients that are negative for CHGA (black) or CHGA, SYP, and NSE (red) yet positive for H19 . B , C , Kaplan–Meir estimates for the impact of H19 on prostate cancer clinical outcomes. B Survival analysis for H19 in ADT untreated patients and C ADT treated patients for biochemical recurrence (BCR—left panels) and metastasis (MET—right panels). Samples were split by H19 tertile expression to test if either of these subgroups of patients were more likely to develop BCR and MET in the context of treatment. P values were calculated using a Kaplan–Meier estimator statistical test to determine significance for the observed stratification in probabilities across the three subgroups in each panel. D , Illustration of H19 transcription and mechanism of action during PCa, NEtD, and tNEPC. During early stage disease, adenocarcinoma cancer cells are driven by an active AR bound to androgens that prevent the transcription of H19 . As the disease progresses (gray triangle), ADTs suppress AR activity, SOX2 increases, and results in the persistent transcription, expression, and activation of H19 . Once active, H19 operates via dual epigenetic mechanisms; (on the left) binds PRC2 complex members, aiding in altering methylation on H3K4Me3/H3K27Me3 histone marks of PRC2 target genes and (on the right) genome-wide alteration of methylation at CpG sites on DNA. Collectively, these epigenetic changes result in the activation of NE gene expression and deactivation of AR signaling gene expression.

    Journal: Nature Communications

    Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

    doi: 10.1038/s41467-021-26901-9

    Figure Lengend Snippet: A Assessment of sensitivity and specificity of H19 in NEPC for use as a diagnostic along with other recently identified NEPC oncogenes (BRN2, SRRM4, and PEG10). Arrows denote patients that are negative for CHGA (black) or CHGA, SYP, and NSE (red) yet positive for H19 . B , C , Kaplan–Meir estimates for the impact of H19 on prostate cancer clinical outcomes. B Survival analysis for H19 in ADT untreated patients and C ADT treated patients for biochemical recurrence (BCR—left panels) and metastasis (MET—right panels). Samples were split by H19 tertile expression to test if either of these subgroups of patients were more likely to develop BCR and MET in the context of treatment. P values were calculated using a Kaplan–Meier estimator statistical test to determine significance for the observed stratification in probabilities across the three subgroups in each panel. D , Illustration of H19 transcription and mechanism of action during PCa, NEtD, and tNEPC. During early stage disease, adenocarcinoma cancer cells are driven by an active AR bound to androgens that prevent the transcription of H19 . As the disease progresses (gray triangle), ADTs suppress AR activity, SOX2 increases, and results in the persistent transcription, expression, and activation of H19 . Once active, H19 operates via dual epigenetic mechanisms; (on the left) binds PRC2 complex members, aiding in altering methylation on H3K4Me3/H3K27Me3 histone marks of PRC2 target genes and (on the right) genome-wide alteration of methylation at CpG sites on DNA. Collectively, these epigenetic changes result in the activation of NE gene expression and deactivation of AR signaling gene expression.

    Article Snippet: Antibodies used: SOX2 (sc-365823, Santa Cruz Biotechnology, 1:400 dilution), H3K27me3 (9733S, Cell Signaling Technology, 1:1000 dilution), EZH2 ((D2C9) XP Rabbit mAb (5246, Cell Signaling Technology, 1:1000 dilution), NSE (sc-21738, Santa Cruz Biotechnology, 1:500 dilution), Synaptophysin (sc-365488, Santa Cruz Biotechnology, 1:500 dilution), CHGA (60893S, Cell Signaling Technology, 1:1000 dilution), BRN2 ((D2C1L) Rabbit mAb 12137, Cell Signaling Technology, 1:1000 dilution), H3 ((D1H2) XP ® Rabbit mAb 4499, Cell Signaling Technology, 1:1000 dilution), Androgen receptor (5153S, Cell Signaling Technology, 1:1000 dilution), H3K4me3 (ab8580, abcam, 1:1000 dilution), P53 (2527 S, Cell Signaling Technology, 1:1000 dilution), Rb (9313S, Cell Signaling Technology, 1:1000 dilution), CK8 (sc–57004, Santa Cruz Biotechnology, 1:300 dilution), PSA (sc-7316, Santa Cruz Biotechnology, 1:250 dilution), HRP conjugated anti-β-actin (Cat. no. A3854, Sigma, 1:10,000 dilution).

    Techniques: Diagnostic Assay, Expressing, Activity Assay, Activation Assay, Methylation, Genome Wide

    Loss of podoplanin restores pigmentation and melanocyte differentiation (A and B) Tumors (A) and cells pellets (B) of PDPN + and PDPN KO B16F10 cell lines. (C) Imaging of pigmentation (brightfield; left) of PDPN + and PDPN KO B16F10 cell lines, labeled with mOrange (red) or CFP (blue) respectively (middle). The scale bar represents 50 microns. (D) Heatmaps showing expression ( Z score) of podoplanin ( PDPN ) and eight dedifferentiation-associated genes in datasets of primary tumor samples of melanoma patients (top; Riker et al. GSE7553 ) and metastatic melanoma cultures (bottom; Mannheim cohort, GSE4843 ; from <xref ref-type=Hoek et al., 2006 ). For the Mannheim cohort, each number indicates a separate metastatic melanoma culture, and the BRAF and/or NRAS mutation status is indicated for each. (E) mRNA expression of five melanocyte-associated ( Mlana/Mart-1 (p = 0.2045), Tyr (p = 0.9276), Dct (p = 0.9859), Gpnmb (p = 0.1308), Pmel (p = 0.9994); left) and three invasion-associated ( Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in PDPN + B16F10 cells. mRNA expression is calculated as fold change of Gapdh expression and normalized to expression in PDPN KO B16F10 cells (set at 1 as indicated by dashed line). Data shown as mean with dots representing n = 3 biological replicates. Two-way ANOVA with Sidak's multiple comparisons; p values indicated above. (F) Western blot analysis of Podoplanin, Melan-A, Tyrosinase and Brn2 (encoded by Pou3f2 gene) in PDPN KO and PDPN + B16F10 cells. Tubulin and histone H3 are used as loading controls. " width="100%" height="100%">

    Journal: iScience

    Article Title: Podoplanin drives dedifferentiation and amoeboid invasion of melanoma

    doi: 10.1016/j.isci.2021.102976

    Figure Lengend Snippet: Loss of podoplanin restores pigmentation and melanocyte differentiation (A and B) Tumors (A) and cells pellets (B) of PDPN + and PDPN KO B16F10 cell lines. (C) Imaging of pigmentation (brightfield; left) of PDPN + and PDPN KO B16F10 cell lines, labeled with mOrange (red) or CFP (blue) respectively (middle). The scale bar represents 50 microns. (D) Heatmaps showing expression ( Z score) of podoplanin ( PDPN ) and eight dedifferentiation-associated genes in datasets of primary tumor samples of melanoma patients (top; Riker et al. GSE7553 ) and metastatic melanoma cultures (bottom; Mannheim cohort, GSE4843 ; from Hoek et al., 2006 ). For the Mannheim cohort, each number indicates a separate metastatic melanoma culture, and the BRAF and/or NRAS mutation status is indicated for each. (E) mRNA expression of five melanocyte-associated ( Mlana/Mart-1 (p = 0.2045), Tyr (p = 0.9276), Dct (p = 0.9859), Gpnmb (p = 0.1308), Pmel (p = 0.9994); left) and three invasion-associated ( Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in PDPN + B16F10 cells. mRNA expression is calculated as fold change of Gapdh expression and normalized to expression in PDPN KO B16F10 cells (set at 1 as indicated by dashed line). Data shown as mean with dots representing n = 3 biological replicates. Two-way ANOVA with Sidak's multiple comparisons; p values indicated above. (F) Western blot analysis of Podoplanin, Melan-A, Tyrosinase and Brn2 (encoded by Pou3f2 gene) in PDPN KO and PDPN + B16F10 cells. Tubulin and histone H3 are used as loading controls.

    Article Snippet: Western blots were incubated with rabbit anti-Phospho-Myosin Light Chain 2 (Thr18/Ser19) (ppMLC; 1:750, Cell Signaling Technology, 3674), rabbit anti-Myosin Light Chain 2 (MLC; 1:750, Cell Signaling Technology, 3672), rabbit anti-MelanA (EPR20380, 1:2000, Abcam, ab210546), mouse anti-human Tyrosinase (T311, 1:100, Santa Cruz, sc-20035), rabbit anti-Brn2/POU3F2 (D2CIL, 1:1000, Cell Signaling Technology, 12137), rabbit anti-podoplanin (1:1000, iSpyBio, ABT34), or as loading controls mouse anti-Tubulin (1:20,000, Sigma Aldrich, T7816), mouse anti-GAPDH (1:10,000, clone 6C5, Millipore, MAB374) or mouse anti-Histone H3 (1:2500, Abcam, ab24824), in PBS supplemented with 1% skim milk powder and 0.2% BSA overnight at 4°C, followed by staining with appropriate HRP-conjugated secondary antibodies (Abcam) for 2 hours at RT.

    Techniques: Imaging, Labeling, Expressing, Mutagenesis, Western Blot