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BioLegend mouse anti tubulin β3
related to Figure 3. Additional Evidence that ZDHHC17 is a Major DLK PAT. A: Quantified intensities of ZDHHC17 protein levels from Fig 3A , normalized to <t>tubulin,</t> confirm efficacy of Zdhhc17 shRNAs. N=9-10 individual cultures per condition; ***:p
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1) Product Images from "Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17"

Article Title: Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17

Journal: bioRxiv

doi: 10.1101/2020.09.01.276287

related to Figure 3. Additional Evidence that ZDHHC17 is a Major DLK PAT. A: Quantified intensities of ZDHHC17 protein levels from Fig 3A , normalized to tubulin, confirm efficacy of Zdhhc17 shRNAs. N=9-10 individual cultures per condition; ***:p
Figure Legend Snippet: related to Figure 3. Additional Evidence that ZDHHC17 is a Major DLK PAT. A: Quantified intensities of ZDHHC17 protein levels from Fig 3A , normalized to tubulin, confirm efficacy of Zdhhc17 shRNAs. N=9-10 individual cultures per condition; ***:p

Techniques Used:

ZDHHC17 Localizes to the Somatic Golgi in Mammalian Sensory Neurons. A: Images of cultured DRG neurons coinfected with lentiviruses to express GFP and HA-ZDHHC17, immunostained with the indicated antibodies. HA-ZDHHC17 is detected in the neuronal soma but not axons. Images are representative of 10 individual neurons examined. Scale bar: 20 μm. B: Western blots of somata plus axons (Soma + Axon) or distal axons only (‘Axon’) fractions from 3 sets of DRG neurons cultured in microfluidic chambers, blotted with the indicated antibodies. The ‘Axon’ chamber was lysed in 1/12 of the volume used for the ‘Soma + Axon’ chamber to account for the lower amount of material in the former compartment. C: Quantified data from n=4 determinations per condition of ZDHHC17 protein level, normalized to tubulin, for each of the indicated subcellular chambers from B . **: p
Figure Legend Snippet: ZDHHC17 Localizes to the Somatic Golgi in Mammalian Sensory Neurons. A: Images of cultured DRG neurons coinfected with lentiviruses to express GFP and HA-ZDHHC17, immunostained with the indicated antibodies. HA-ZDHHC17 is detected in the neuronal soma but not axons. Images are representative of 10 individual neurons examined. Scale bar: 20 μm. B: Western blots of somata plus axons (Soma + Axon) or distal axons only (‘Axon’) fractions from 3 sets of DRG neurons cultured in microfluidic chambers, blotted with the indicated antibodies. The ‘Axon’ chamber was lysed in 1/12 of the volume used for the ‘Soma + Axon’ chamber to account for the lower amount of material in the former compartment. C: Quantified data from n=4 determinations per condition of ZDHHC17 protein level, normalized to tubulin, for each of the indicated subcellular chambers from B . **: p

Techniques Used: Cell Culture, Western Blot

2) Product Images from "An Autism-Associated Mutation Impairs Neuroligin-4 Glycosylation and Enhances Excitatory Synaptic Transmission in Human Neurons"

Article Title: An Autism-Associated Mutation Impairs Neuroligin-4 Glycosylation and Enhances Excitatory Synaptic Transmission in Human Neurons

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0404-20.2020

R101Q mutation inhibits NLGN4-induced excitatory synapse formation in NSC-derived human neurons. A , NSCs were induced from H1-ES cells by dual-SMAD inhibition. The NSC-derived neurons were cocultured with glia, infected with lentivirus-expressing transgenes, and analyzed at indicated time points (arrowheads). B , Design of lentiviral vectors expressing NLGN4 WT or R101Q variant under hSyn1 promoter followed by an IRES-EGFP construct. An EGFP-only vector was used as infection control (Ctrl). C , Images illustrate cellular identities, when immunolabeled for ES cell marker (Oct3/4), NSC marker (Nestin), or neuronal marker (Dcx), at different stages of the differentiation protocol. DAPI was used for nuclear stain. At day 60, elaborate neuronal morphology was confirmed by EGFP signal. Arrowheads point at neurons (yellow) and glial cells (white) in the culture. D , Sample images (top) depict NSC-derived neurons expressing NLGN4 WT versus R101Q variant, when immunostained for cytoskeletal marker Tuj1 and synaptic marker Synapsin. Bar graphs (below) represent normalized counts of cell bodies per field of view (left), total neurite volume (middle), and integrated signal intensity of Synapsin (right), for WT versus R101Q conditions. E , Representative images (top) of MAP2-positive dendritic segments counterstained for Synapsin, and summary graphs (bottom) of the density and size of Synapsin puncta, as quantified from cells expressing NLGN4 WT versus R101Q mutant. F , Example images (left) of MAP2-positive dendritic branches from cells in control, NLGN4 WT, and R101Q mutant conditions, as counterstained for an inhibitory synapse marker vGAT. Summary graphs (right) of the density and size of vGAT puncta. G , Same as F , except the neurons were immunolabeled for VGluT, an excitatory synapse marker. Averages indicate the mean ± SEM, with the total number of fields of view analyzed/number of batches. Statistical significance was tested by two-tailed, unpaired, Student's t test. *** p
Figure Legend Snippet: R101Q mutation inhibits NLGN4-induced excitatory synapse formation in NSC-derived human neurons. A , NSCs were induced from H1-ES cells by dual-SMAD inhibition. The NSC-derived neurons were cocultured with glia, infected with lentivirus-expressing transgenes, and analyzed at indicated time points (arrowheads). B , Design of lentiviral vectors expressing NLGN4 WT or R101Q variant under hSyn1 promoter followed by an IRES-EGFP construct. An EGFP-only vector was used as infection control (Ctrl). C , Images illustrate cellular identities, when immunolabeled for ES cell marker (Oct3/4), NSC marker (Nestin), or neuronal marker (Dcx), at different stages of the differentiation protocol. DAPI was used for nuclear stain. At day 60, elaborate neuronal morphology was confirmed by EGFP signal. Arrowheads point at neurons (yellow) and glial cells (white) in the culture. D , Sample images (top) depict NSC-derived neurons expressing NLGN4 WT versus R101Q variant, when immunostained for cytoskeletal marker Tuj1 and synaptic marker Synapsin. Bar graphs (below) represent normalized counts of cell bodies per field of view (left), total neurite volume (middle), and integrated signal intensity of Synapsin (right), for WT versus R101Q conditions. E , Representative images (top) of MAP2-positive dendritic segments counterstained for Synapsin, and summary graphs (bottom) of the density and size of Synapsin puncta, as quantified from cells expressing NLGN4 WT versus R101Q mutant. F , Example images (left) of MAP2-positive dendritic branches from cells in control, NLGN4 WT, and R101Q mutant conditions, as counterstained for an inhibitory synapse marker vGAT. Summary graphs (right) of the density and size of vGAT puncta. G , Same as F , except the neurons were immunolabeled for VGluT, an excitatory synapse marker. Averages indicate the mean ± SEM, with the total number of fields of view analyzed/number of batches. Statistical significance was tested by two-tailed, unpaired, Student's t test. *** p

Techniques Used: Mutagenesis, Derivative Assay, Inhibition, Infection, Expressing, Variant Assay, Construct, Plasmid Preparation, Immunolabeling, Marker, Staining, Two Tailed Test

3) Product Images from "Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia"

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

Journal: Nature Communications

doi: 10.1038/s41467-021-25344-6

Human iPSC-derived neuronal model of Alzheimer’s disease recapitulates key hallmark AD pathologies. a , b Differentiated NAG neurons (12 weeks+) show loss of dendrites (MAP2, green) and cell bodies (CUX2, red) when treated with soluble Aβ species for 7 days ( b ) in comparison to no treatment condition ( a ). c Anti-Aβ antibody co-treatment with soluble Aβ species blocks Aβ-induced cell death. Scale bar 50 μm. d Dose-dependent, progressive cell death as quantified by the percentage of cell body (CUX2) numbers in Aβ-treated normalized to untreated control. e Dose-dependent, progressive dendritic (MAP2) loss as quantified by the percentage of MAP2 area in Aβ-treated normalized to untreated control. f , g Aβ42 treatment induces phosphorylation of Tau (p-Tau 396–404, white) and mislocalization to the cell body. h Anti-Aβ antibody co-treatment with sAβ42s blocks Aβ-induced Tau hyperphosphorylation. Scale bar 50 μm. i Dose-dependent and time course of phosphorylation of Tau at S396/404. Phospho-Tau induced increase at 5 µM Aβ treatment before decrease associated with cell death occurred as quantified by fold p-Tau 396/404 staining in Aβ-treated normalized to untreated control. j , k Aβ42 treatment causes synapse loss in neurons (synapsin, green). l Anti-Aβ antibody co-treatment with sAβ42s blocks synapse loss phenotype. Scale bar = 5 μm. m Dose–response and time course of synapse (synapsin 1/2) loss in Aβ-treated culture normalized to untreated control. n , o sAβ42s treatment induces axon fragmentation (beta-3 tubulin Tuj1, white). p Anti-Aβ antibody co-treatment blocks axon fragmentation. Scale bar = 50 μm. q Dose–response and time course of axon fragmentation as quantified by percentage of the axon (NFL-H) area in Aβ-treated normalized to untreated control. r Anti-Aβ antibody rescues all three markers in a dose-dependent manner and IC50 curves can be drawn and calculated (IC50 curve fitted by Prism software). Data are presented as mean values +/− SEM and n = 4 wells. All scale bars = 50 μm.
Figure Legend Snippet: Human iPSC-derived neuronal model of Alzheimer’s disease recapitulates key hallmark AD pathologies. a , b Differentiated NAG neurons (12 weeks+) show loss of dendrites (MAP2, green) and cell bodies (CUX2, red) when treated with soluble Aβ species for 7 days ( b ) in comparison to no treatment condition ( a ). c Anti-Aβ antibody co-treatment with soluble Aβ species blocks Aβ-induced cell death. Scale bar 50 μm. d Dose-dependent, progressive cell death as quantified by the percentage of cell body (CUX2) numbers in Aβ-treated normalized to untreated control. e Dose-dependent, progressive dendritic (MAP2) loss as quantified by the percentage of MAP2 area in Aβ-treated normalized to untreated control. f , g Aβ42 treatment induces phosphorylation of Tau (p-Tau 396–404, white) and mislocalization to the cell body. h Anti-Aβ antibody co-treatment with sAβ42s blocks Aβ-induced Tau hyperphosphorylation. Scale bar 50 μm. i Dose-dependent and time course of phosphorylation of Tau at S396/404. Phospho-Tau induced increase at 5 µM Aβ treatment before decrease associated with cell death occurred as quantified by fold p-Tau 396/404 staining in Aβ-treated normalized to untreated control. j , k Aβ42 treatment causes synapse loss in neurons (synapsin, green). l Anti-Aβ antibody co-treatment with sAβ42s blocks synapse loss phenotype. Scale bar = 5 μm. m Dose–response and time course of synapse (synapsin 1/2) loss in Aβ-treated culture normalized to untreated control. n , o sAβ42s treatment induces axon fragmentation (beta-3 tubulin Tuj1, white). p Anti-Aβ antibody co-treatment blocks axon fragmentation. Scale bar = 50 μm. q Dose–response and time course of axon fragmentation as quantified by percentage of the axon (NFL-H) area in Aβ-treated normalized to untreated control. r Anti-Aβ antibody rescues all three markers in a dose-dependent manner and IC50 curves can be drawn and calculated (IC50 curve fitted by Prism software). Data are presented as mean values +/− SEM and n = 4 wells. All scale bars = 50 μm.

Techniques Used: Derivative Assay, Staining, Software

4) Product Images from "3D-imaging reveals conserved cerebrospinal fluid drainage via meningeal lymphatic vasculature in mice and humans"

Article Title: 3D-imaging reveals conserved cerebrospinal fluid drainage via meningeal lymphatic vasculature in mice and humans

Journal: bioRxiv

doi: 10.1101/2022.01.13.476230

Dorsal and basal meningeal lymphatic drainage. A LSFM lateral view of OVA-A 555 (magenta) in the posterior head after anti-LYVE1 antibody labeling (green). The animal was sacrificed 45 min after Lb-Sa OVA-A 555 injection. OVA-A 555 is concentrated in brain meninges, within the perivascular spaces along the spinal cord (SC), the cerebellum (Cb) and the cortex (Cx). Outside of the ventral skull border (dashed line), OVA-A 555 is detected in the dcLN. NP: nasopharyngeal cavity (solid line). Dashed rectangles correspond to regions magnified in the indicated panels, MLVs and tracer uptake region are numbered in green (I-IV). Inset in A shows a schematic of dural veins and sinuses (light and dark blue), the light blue veins are the focus of this figure: cav: cavenous sinus, iptgv: interpterygoid emmissary vein, jugv: jugular vein, pfv: posterior facial vein, PSS: petrosquamous sinus, rgv: retroglenoid vein, SS: sigmoid sinus, SSS: superior sagittal sinus, TS: transverse sinus. B-E LSFM horizontal ( B, C ) and sagittal views ( D, E ) of the calvaria, dorsal sinuses are stained with vWF antibody ( B, C ) and MLVs with LYVE antibody ( D , E ). The SSS connects the RCS with the COS ( B , blue arrowheads) which separates in two branches forming the TS ( C ). Note discontinuous OVA-A 555 labeling in the perisinus spaces ( B , C ). LYVE-1+ MLVs line the SSS and TS ( D, I) and contain OVA-A 555 at the TS ( E, II), at the COS and the RCS ( D ), blue arrowheads. F-H LSFM coronal views at the level of the foramen of the jugular vein labeled with the indicated antibodies and OVA-A 555 . vWF stained the SS and the jugv, OVA-A 555 is accumulated around the jugular foramen. ( G ) TUJ1 stained nerves IX, X and XI, which cross the skull through the jugular foramen, OVA-A 555 is close but not associated directly with the nerves. ( H ) LYVE1 + MLVs follow the SS until the jugular vein exits the skull by the jugular foramen. Some MLVs transport OVA-A 555 positive phagocytic cells (III). IX : cranial nerve 9 (Glossopharyngeal), X : cranial nerve 10 (Vagus), X : cranial nerve 11 (Spinal accessory). I-K LSFM coronal views of the petrosquamous sinus exit through the skull.( i ) vWF stained the PSS passing through the petrosquamous sinus foramen (white arrow) to join the pfv via the rgv, OVA-A 555 is accumulated around the jugular foramen. ( J , K ) LYVE1 + MLVs follow the PSS until the rgv and the pfv. Some MLVs show OVA-A 555 + phagocytic cells at the petrosquamous fissure level (blue arrowhead white arrow?). Scale bar: 1000 μm ( A - D ); 500 μm ( E - J ); 250 μm ( K ).
Figure Legend Snippet: Dorsal and basal meningeal lymphatic drainage. A LSFM lateral view of OVA-A 555 (magenta) in the posterior head after anti-LYVE1 antibody labeling (green). The animal was sacrificed 45 min after Lb-Sa OVA-A 555 injection. OVA-A 555 is concentrated in brain meninges, within the perivascular spaces along the spinal cord (SC), the cerebellum (Cb) and the cortex (Cx). Outside of the ventral skull border (dashed line), OVA-A 555 is detected in the dcLN. NP: nasopharyngeal cavity (solid line). Dashed rectangles correspond to regions magnified in the indicated panels, MLVs and tracer uptake region are numbered in green (I-IV). Inset in A shows a schematic of dural veins and sinuses (light and dark blue), the light blue veins are the focus of this figure: cav: cavenous sinus, iptgv: interpterygoid emmissary vein, jugv: jugular vein, pfv: posterior facial vein, PSS: petrosquamous sinus, rgv: retroglenoid vein, SS: sigmoid sinus, SSS: superior sagittal sinus, TS: transverse sinus. B-E LSFM horizontal ( B, C ) and sagittal views ( D, E ) of the calvaria, dorsal sinuses are stained with vWF antibody ( B, C ) and MLVs with LYVE antibody ( D , E ). The SSS connects the RCS with the COS ( B , blue arrowheads) which separates in two branches forming the TS ( C ). Note discontinuous OVA-A 555 labeling in the perisinus spaces ( B , C ). LYVE-1+ MLVs line the SSS and TS ( D, I) and contain OVA-A 555 at the TS ( E, II), at the COS and the RCS ( D ), blue arrowheads. F-H LSFM coronal views at the level of the foramen of the jugular vein labeled with the indicated antibodies and OVA-A 555 . vWF stained the SS and the jugv, OVA-A 555 is accumulated around the jugular foramen. ( G ) TUJ1 stained nerves IX, X and XI, which cross the skull through the jugular foramen, OVA-A 555 is close but not associated directly with the nerves. ( H ) LYVE1 + MLVs follow the SS until the jugular vein exits the skull by the jugular foramen. Some MLVs transport OVA-A 555 positive phagocytic cells (III). IX : cranial nerve 9 (Glossopharyngeal), X : cranial nerve 10 (Vagus), X : cranial nerve 11 (Spinal accessory). I-K LSFM coronal views of the petrosquamous sinus exit through the skull.( i ) vWF stained the PSS passing through the petrosquamous sinus foramen (white arrow) to join the pfv via the rgv, OVA-A 555 is accumulated around the jugular foramen. ( J , K ) LYVE1 + MLVs follow the PSS until the rgv and the pfv. Some MLVs show OVA-A 555 + phagocytic cells at the petrosquamous fissure level (blue arrowhead white arrow?). Scale bar: 1000 μm ( A - D ); 500 μm ( E - J ); 250 μm ( K ).

Techniques Used: Antibody Labeling, Injection, Staining, Labeling

LSFM 3D-images of MLVs associated with the caudal cavernous sinuses in the middle fossa of the skull base. A Schematic of the venous sinuses, veins and internal carotid artery at the base of the brain. Stippled areas correspond to the 3 coronal panels (1-3) shown in ( C , F ) for 1, ( E , H ) for 2 and ( I , J ) for 3. Inset in A : coronal section plane of the head used in B - K . bv: basilar venous plexus, cav: cavernous sinus, ic: internal carotid artery, icav: inter-cavernous sinus, ipets: inferior petrosal sinus, iptgev: interpterygoid emmissary vein, jugv: internal jugular vein, OB: olfactory bulb, pbv: posterior basal vein, pfv: posterior facial vein, spets: superior petrosal sinus. B OVA-A 555 was injected into Th-Lb 45 min before sacrifice and sample was labeled with the indicated antibodies. Blood vessels of the pituitary gland (PG) and dura mater express CD31 and PDLX (blue arrowheads). Tracer deposits (magenta) were observed in the pia mater of the brain, around the cavernous sinus (cav) and the posterior basal vein (pbv). C-E Ventral ( C , D ) and dorsal ( E ) views of a sample stained with vWF to label dural sinuses and veins (blue arrowheads). TS: transverse sinus. Tracer accumulated in peri-venous and -sinusal spaces, especially at confluence points. Dashed line: limit between intracranial and extracranial regions. F-H Lymphatic vasculature (LYVE1 + , green in F , G ) and TUJ1 + cranial nerves (yellow in G ), VI : cranial nerve 6 (Abducens) at coronal levels 1 and 2. MLVs contacted the cavernous perisinus space where tracer accumulated ( F ), and surrounded the foramen of iptgev ( H ). Cranial nerves were devoid of tracer deposits and MLVs. I-L Dural veins (vWF, blue in I ), lymphatic vasculature (LYVE1 + , green in K , L ) and cranial nerves (TUJ1 + , yellow in J ) on coronal ( I-K ) and sagittal ( L ) views at level 3. MLVs contacted the cavernous sinuses ( K , L ) and lymphatic tracer uptake (white in K ) was detected at the intersection of cavernous sinuses with internal carotid arteries in the skull base. Cranial nerves showed neither tracer deposits nor MLVs ( J ). V : cranial nerve 5 (Trigeminal), IV : cranial nerve 4 (Trochlear), III : cranial nerve 3 (Oculomotor). Br: brain, CC: carotid canal, NP: nasopharyngeal cavity. A: anterior, D: dorsal, L: lateral, P: posterior, V: ventral. Scale bar: 500 μm ( B - L ).
Figure Legend Snippet: LSFM 3D-images of MLVs associated with the caudal cavernous sinuses in the middle fossa of the skull base. A Schematic of the venous sinuses, veins and internal carotid artery at the base of the brain. Stippled areas correspond to the 3 coronal panels (1-3) shown in ( C , F ) for 1, ( E , H ) for 2 and ( I , J ) for 3. Inset in A : coronal section plane of the head used in B - K . bv: basilar venous plexus, cav: cavernous sinus, ic: internal carotid artery, icav: inter-cavernous sinus, ipets: inferior petrosal sinus, iptgev: interpterygoid emmissary vein, jugv: internal jugular vein, OB: olfactory bulb, pbv: posterior basal vein, pfv: posterior facial vein, spets: superior petrosal sinus. B OVA-A 555 was injected into Th-Lb 45 min before sacrifice and sample was labeled with the indicated antibodies. Blood vessels of the pituitary gland (PG) and dura mater express CD31 and PDLX (blue arrowheads). Tracer deposits (magenta) were observed in the pia mater of the brain, around the cavernous sinus (cav) and the posterior basal vein (pbv). C-E Ventral ( C , D ) and dorsal ( E ) views of a sample stained with vWF to label dural sinuses and veins (blue arrowheads). TS: transverse sinus. Tracer accumulated in peri-venous and -sinusal spaces, especially at confluence points. Dashed line: limit between intracranial and extracranial regions. F-H Lymphatic vasculature (LYVE1 + , green in F , G ) and TUJ1 + cranial nerves (yellow in G ), VI : cranial nerve 6 (Abducens) at coronal levels 1 and 2. MLVs contacted the cavernous perisinus space where tracer accumulated ( F ), and surrounded the foramen of iptgev ( H ). Cranial nerves were devoid of tracer deposits and MLVs. I-L Dural veins (vWF, blue in I ), lymphatic vasculature (LYVE1 + , green in K , L ) and cranial nerves (TUJ1 + , yellow in J ) on coronal ( I-K ) and sagittal ( L ) views at level 3. MLVs contacted the cavernous sinuses ( K , L ) and lymphatic tracer uptake (white in K ) was detected at the intersection of cavernous sinuses with internal carotid arteries in the skull base. Cranial nerves showed neither tracer deposits nor MLVs ( J ). V : cranial nerve 5 (Trigeminal), IV : cranial nerve 4 (Trochlear), III : cranial nerve 3 (Oculomotor). Br: brain, CC: carotid canal, NP: nasopharyngeal cavity. A: anterior, D: dorsal, L: lateral, P: posterior, V: ventral. Scale bar: 500 μm ( B - L ).

Techniques Used: Injection, Labeling, Staining

5) Product Images from "Novel role of Lin28 signaling in regulation of mammalian PNS and CNS axon regeneration"

Article Title: Novel role of Lin28 signaling in regulation of mammalian PNS and CNS axon regeneration

Journal: bioRxiv

doi: 10.1101/281725

Overexpression of Lin28a did not affect survival rate of RGCs, while optic nerve crush caused continuous cell death. Related to Figure 5 . (A) Representative images of whole-mount retinas showing that overexpression of Lin28a in retinas had no effect on RGC survival rate, while fewer RGCs survived for 4 weeks after optic nerve crush compared to 2 weeks after optic nerve crush. Whole-mount retinas were stained with anti-βIII tubulin antibody (Tuj1, green). Scale bar, 50 µm. (B) Quantification of survival rate of RGCs in (A) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0062, n = 3 mice in each group). n.s., not significant. ** P
Figure Legend Snippet: Overexpression of Lin28a did not affect survival rate of RGCs, while optic nerve crush caused continuous cell death. Related to Figure 5 . (A) Representative images of whole-mount retinas showing that overexpression of Lin28a in retinas had no effect on RGC survival rate, while fewer RGCs survived for 4 weeks after optic nerve crush compared to 2 weeks after optic nerve crush. Whole-mount retinas were stained with anti-βIII tubulin antibody (Tuj1, green). Scale bar, 50 µm. (B) Quantification of survival rate of RGCs in (A) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0062, n = 3 mice in each group). n.s., not significant. ** P

Techniques Used: Over Expression, Staining, Mouse Assay

Downregulation of let-7 miRNAs was required for sensory axon regeneration in vitro and in vivo. (A) let-7a (left) and let-7b (right) levels were significantly decreased in L4/5 DRGs 7 days after sciatic nerve injury (SNI) (one sample t test, P = 0.0004 for let-7a, P = 0.0010 for let-7b, n = 8 independent experiments). (B) let-7a (left) or let-7b (right) levels in cultured DRG neurons were significantly increased 3 days after let-7a or let-7b mimic electroporation (one sample t test, P = 0.0346 for let-7a, P = 0.0205 for let-7b, n = 3 independent experiments). (C) Representative images of cultured DRG neurons showing that overexpression of let-7a or let-7b impaired sensory axon regeneration in vitro. Cells were stained with anti-GFP (green) and anti-βIII tubulin (Tuj1, red) antibodies. Only axons of GFP + /Tuj1 + cells were measured. Scale bar, 100µm. (D) Top: diagram showing the timeline of the experiment. Bottom: representative images of regenerating sciatic nerves showing that overexpression of let-7a impaired sensory axon regeneration in vivo. The right column shows enlarged images of areas in the dashed white boxes in the left column. The red line indicates the crush sites. Red arrows indicate distal tips of regenerating axons. Scale bar, 1 mm for the left column, 0.5 mm for the right column. (E) Quantification of average axon length in (C) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0002, n = 3 independent experiments). Quantification of average axon length in (D) (unpaired Student’s t test, P = 0.0175, n = 6 mice in each group). (F) let-7a level in L4/5 DRGs was significantly increased 2 days after in vivo electroporation of let-7a mimic (one sample t test, P = 0.0023, n = 3 independent experiments). * P
Figure Legend Snippet: Downregulation of let-7 miRNAs was required for sensory axon regeneration in vitro and in vivo. (A) let-7a (left) and let-7b (right) levels were significantly decreased in L4/5 DRGs 7 days after sciatic nerve injury (SNI) (one sample t test, P = 0.0004 for let-7a, P = 0.0010 for let-7b, n = 8 independent experiments). (B) let-7a (left) or let-7b (right) levels in cultured DRG neurons were significantly increased 3 days after let-7a or let-7b mimic electroporation (one sample t test, P = 0.0346 for let-7a, P = 0.0205 for let-7b, n = 3 independent experiments). (C) Representative images of cultured DRG neurons showing that overexpression of let-7a or let-7b impaired sensory axon regeneration in vitro. Cells were stained with anti-GFP (green) and anti-βIII tubulin (Tuj1, red) antibodies. Only axons of GFP + /Tuj1 + cells were measured. Scale bar, 100µm. (D) Top: diagram showing the timeline of the experiment. Bottom: representative images of regenerating sciatic nerves showing that overexpression of let-7a impaired sensory axon regeneration in vivo. The right column shows enlarged images of areas in the dashed white boxes in the left column. The red line indicates the crush sites. Red arrows indicate distal tips of regenerating axons. Scale bar, 1 mm for the left column, 0.5 mm for the right column. (E) Quantification of average axon length in (C) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0002, n = 3 independent experiments). Quantification of average axon length in (D) (unpaired Student’s t test, P = 0.0175, n = 6 mice in each group). (F) let-7a level in L4/5 DRGs was significantly increased 2 days after in vivo electroporation of let-7a mimic (one sample t test, P = 0.0023, n = 3 independent experiments). * P

Techniques Used: In Vitro, In Vivo, Cell Culture, Electroporation, Over Expression, Staining, Mouse Assay

Upregulation of Lin28a and Lin28b was required for sensory axon regeneration in vitro and in vivo. (A) Lin28a (left) and Lin28b (right) mRNA levels were significantly increased in L4/5 DRGs 7 days after sciatic nerve injury (SNI) (one sample t test, P = 0.0039 for Lin28a, P = 0.0003 for Lin28b, n = 13 independent experiments). (B) Representative western blot results (2 out of 3 independent experiments) showing markedly increased Lin28a protein level in L4/5 DRGs 1 day or 3 days after SNI. (C) Quantification of relative Lin28a protein level in (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0211, n = 3 independent experiments). (D) Representative images of cultured DRG neurons showing that simultaneously knocking down Lin28a and Lin28b impaired sensory axon regeneration in vitro. Cells were stained with anti-GFP (green) and anti-βIII tubulin (Tuj1, red) antibodies. Only axons of GFP + /Tuj1 + cells were measured. Scale bar, 100 µm. (E) Quantification of average axon length in (D) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0009, n = 3 independent experiments). (F) Top: diagram showing the timeline of the experiment. Bottom: representative images of regenerating sciatic nerves showing that simultaneously knocking down Lin28a and Lin28b impaired sensory axon regeneration in vivo. The right column shows enlarged images of areas in the dashed white boxes in the left column. The red line indicates the crush sites. Red arrows indicate distal tips of regenerating axons. Scale bar, 1 mm for the left column, 0.5 mm for the right column. (G) Quantification of average axon length in (F) (unpaired Student’s t test, P = 0.0064, n = 6 mice in each group). * P
Figure Legend Snippet: Upregulation of Lin28a and Lin28b was required for sensory axon regeneration in vitro and in vivo. (A) Lin28a (left) and Lin28b (right) mRNA levels were significantly increased in L4/5 DRGs 7 days after sciatic nerve injury (SNI) (one sample t test, P = 0.0039 for Lin28a, P = 0.0003 for Lin28b, n = 13 independent experiments). (B) Representative western blot results (2 out of 3 independent experiments) showing markedly increased Lin28a protein level in L4/5 DRGs 1 day or 3 days after SNI. (C) Quantification of relative Lin28a protein level in (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0211, n = 3 independent experiments). (D) Representative images of cultured DRG neurons showing that simultaneously knocking down Lin28a and Lin28b impaired sensory axon regeneration in vitro. Cells were stained with anti-GFP (green) and anti-βIII tubulin (Tuj1, red) antibodies. Only axons of GFP + /Tuj1 + cells were measured. Scale bar, 100 µm. (E) Quantification of average axon length in (D) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0009, n = 3 independent experiments). (F) Top: diagram showing the timeline of the experiment. Bottom: representative images of regenerating sciatic nerves showing that simultaneously knocking down Lin28a and Lin28b impaired sensory axon regeneration in vivo. The right column shows enlarged images of areas in the dashed white boxes in the left column. The red line indicates the crush sites. Red arrows indicate distal tips of regenerating axons. Scale bar, 1 mm for the left column, 0.5 mm for the right column. (G) Quantification of average axon length in (F) (unpaired Student’s t test, P = 0.0064, n = 6 mice in each group). * P

Techniques Used: In Vitro, In Vivo, Western Blot, Cell Culture, Staining, Mouse Assay

Most RGCs were transduced with AAV2-Lin28a-Flag 2 weeks after viral injection. Related to Figure 5 . (A) Representative images of whole-mount retinas showing the high transduction rate of AAV2-Lin28a-Flag in RGCs. Whole-mount retinas were stained with anti-βIII tubulin (Tuj1, red) and anti-Flag (green) antibodies. Scale bar, 1 mm for the upper row, 50 µm for the lower row. (B) Quantification of the transduction rate of AAV2-Lin28a-Flag in RGCs in (A). The average transduction rate was 87.87 ± 5.084%. Each dot represents an independently injected retina (n = 3 retinas). Data are presented as mean ± SD.
Figure Legend Snippet: Most RGCs were transduced with AAV2-Lin28a-Flag 2 weeks after viral injection. Related to Figure 5 . (A) Representative images of whole-mount retinas showing the high transduction rate of AAV2-Lin28a-Flag in RGCs. Whole-mount retinas were stained with anti-βIII tubulin (Tuj1, red) and anti-Flag (green) antibodies. Scale bar, 1 mm for the upper row, 50 µm for the lower row. (B) Quantification of the transduction rate of AAV2-Lin28a-Flag in RGCs in (A). The average transduction rate was 87.87 ± 5.084%. Each dot represents an independently injected retina (n = 3 retinas). Data are presented as mean ± SD.

Techniques Used: Transduction, Injection, Staining

Lin28 overexpression in sensory neurons or RGCs was associated with Akt, GSK3β, and mTOR signaling. (A) Western blot results of 3 independent experiments showing overexpression of hLin28b elevated levels of pAkt, pGSK3β and pS6 in L4/5 DRGs. (B) Quantification of relative pAkt, pGSK3β and pS6 levels in (A) (unpaired Student’s t test, P = 0.0372, 0.0217 and 0.0453 for pAkt, pGSK3β and pS6, respectively, n = 3 independent experiments). (C) Representative images of retinal cyrosections showing that overexpression of Lin28a in retinas markedly increased the percentage of pS6 + RGCs in RGC layer. Cryosections were stained with anti-pS6 (green) and anti-βIII tubulin (Tuj1, far-red) antibodies. White arrows indicate pS6 + RGCs. Scale bar, 50 µm. (D) Quantification of percentage of pS6 + RGCs in RGC layer of control retinas and Lin28a overexpression retinas (unpaired Student’s t test, P = 0.0027, n = 3 mice in control group, n = 4 mice in Lin28a overexpression group). * P
Figure Legend Snippet: Lin28 overexpression in sensory neurons or RGCs was associated with Akt, GSK3β, and mTOR signaling. (A) Western blot results of 3 independent experiments showing overexpression of hLin28b elevated levels of pAkt, pGSK3β and pS6 in L4/5 DRGs. (B) Quantification of relative pAkt, pGSK3β and pS6 levels in (A) (unpaired Student’s t test, P = 0.0372, 0.0217 and 0.0453 for pAkt, pGSK3β and pS6, respectively, n = 3 independent experiments). (C) Representative images of retinal cyrosections showing that overexpression of Lin28a in retinas markedly increased the percentage of pS6 + RGCs in RGC layer. Cryosections were stained with anti-pS6 (green) and anti-βIII tubulin (Tuj1, far-red) antibodies. White arrows indicate pS6 + RGCs. Scale bar, 50 µm. (D) Quantification of percentage of pS6 + RGCs in RGC layer of control retinas and Lin28a overexpression retinas (unpaired Student’s t test, P = 0.0027, n = 3 mice in control group, n = 4 mice in Lin28a overexpression group). * P

Techniques Used: Over Expression, Western Blot, Staining, Mouse Assay

6) Product Images from "Eomes and Brachyury control pluripotency exit and germ layer segregation by changes of chromatin state"

Article Title: Eomes and Brachyury control pluripotency exit and germ layer segregation by changes of chromatin state

Journal: bioRxiv

doi: 10.1101/774232

Characterization of dKO cells for EOMES and BRACHYURY expression, reporter activation, and differentiation potential. a , Schematic of Eomes Gfp/Δ (EoKO) and Bra Tom/Δ (BraKO) alleles. Fluorescence reporters Gfp and Tom, including polyA signals were inserted into the start codons of one allele of Eomes and Brachyury , respectively, and the second allele is functionally disrupted using TALENs to generate short out-of-frame deletions within the first exon. b , Relative mRNA expression of mesoderm and endoderm (ME) genes alongside with Eomes and Brachyury expression over 5 days of differentiation of WT cells. Error bars represent SEM. c , Expression levels of Eomes and Brachyury transcripts during the 5 days of differentiation of WT, BraKO, EoKO, and dKO cells measured by qRT-PCR. Error bars indicate SEM; p-values for differences of mean expression between WT and dKO samples were calculated by Student’s t-test. n.s:p > 0.05; *:p≤0.05; **:p≤0.01; ***: p≤0.001; ****: p≤0.0001. d , Immunofluorescence staining at day 4 of differentiation demonstrating the absence of EOMES and BRACHYURY in respective loss-of-function cell lines. Scale bars 100 μm. e , Eomes Gfp and Bra Tom reporter activation in Eomes Gfp/+ and dKO EBs at day 4 of differentiation. Maximum intensity projection of confocal z-stacks is shown. Scale bars 100 μm. f , Protein levels of non-phosphorylated (active) β-CATENIN and total β-CATENIN in WT and dKO cells showing responsiveness to WNT stimulation. β-ACTIN served as the loading control. g , Protein levels of phosphorylated SMAD2 and total SMAD2 in WT and dKO cells showing responsiveness to ActA stimulation. ɑ-TUBULIN served as the loading control. h , Super 8x TOPflash luciferase reporter assay demonstrating responsiveness of WT and dKO cells to WNT stimulation when treated with WNT3A L-cell conditioned medium (CM), but not when untreated or inhibited with XAV939. Error bars indicate SEM. p-values for differences of mean expression between treated and untreated samples were calculated by Student’s t-test. *:p≤0.05; **:p≤ 0.01 in h and i. i , 6xARE Luciferase reporter assay demonstrating responsiveness of WT and dKO cells to TGFβ/Nodal signalling when treated with ActA, but not when untreated or inhibited with SB431542. j , Immunofluorescence staining for SOX17, FIBRONECTIN1 (FN1), FOXA2, and FOXC2 proteins in plated EBs at day 4 and 7 of differentiation showing the absence of endoderm (SOX17 and FOXA2) and mesoderm (FN1 and FOXC2) markers. Scale bars 100 μm.
Figure Legend Snippet: Characterization of dKO cells for EOMES and BRACHYURY expression, reporter activation, and differentiation potential. a , Schematic of Eomes Gfp/Δ (EoKO) and Bra Tom/Δ (BraKO) alleles. Fluorescence reporters Gfp and Tom, including polyA signals were inserted into the start codons of one allele of Eomes and Brachyury , respectively, and the second allele is functionally disrupted using TALENs to generate short out-of-frame deletions within the first exon. b , Relative mRNA expression of mesoderm and endoderm (ME) genes alongside with Eomes and Brachyury expression over 5 days of differentiation of WT cells. Error bars represent SEM. c , Expression levels of Eomes and Brachyury transcripts during the 5 days of differentiation of WT, BraKO, EoKO, and dKO cells measured by qRT-PCR. Error bars indicate SEM; p-values for differences of mean expression between WT and dKO samples were calculated by Student’s t-test. n.s:p > 0.05; *:p≤0.05; **:p≤0.01; ***: p≤0.001; ****: p≤0.0001. d , Immunofluorescence staining at day 4 of differentiation demonstrating the absence of EOMES and BRACHYURY in respective loss-of-function cell lines. Scale bars 100 μm. e , Eomes Gfp and Bra Tom reporter activation in Eomes Gfp/+ and dKO EBs at day 4 of differentiation. Maximum intensity projection of confocal z-stacks is shown. Scale bars 100 μm. f , Protein levels of non-phosphorylated (active) β-CATENIN and total β-CATENIN in WT and dKO cells showing responsiveness to WNT stimulation. β-ACTIN served as the loading control. g , Protein levels of phosphorylated SMAD2 and total SMAD2 in WT and dKO cells showing responsiveness to ActA stimulation. ɑ-TUBULIN served as the loading control. h , Super 8x TOPflash luciferase reporter assay demonstrating responsiveness of WT and dKO cells to WNT stimulation when treated with WNT3A L-cell conditioned medium (CM), but not when untreated or inhibited with XAV939. Error bars indicate SEM. p-values for differences of mean expression between treated and untreated samples were calculated by Student’s t-test. *:p≤0.05; **:p≤ 0.01 in h and i. i , 6xARE Luciferase reporter assay demonstrating responsiveness of WT and dKO cells to TGFβ/Nodal signalling when treated with ActA, but not when untreated or inhibited with SB431542. j , Immunofluorescence staining for SOX17, FIBRONECTIN1 (FN1), FOXA2, and FOXC2 proteins in plated EBs at day 4 and 7 of differentiation showing the absence of endoderm (SOX17 and FOXA2) and mesoderm (FN1 and FOXC2) markers. Scale bars 100 μm.

Techniques Used: Expressing, Activation Assay, Fluorescence, TALENs, Quantitative RT-PCR, Immunofluorescence, Staining, Luciferase, Reporter Assay

7) Product Images from "Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models"

Article Title: Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2022.06.015

TECPR2 and TECPR2ΔExon8 neuronal localization in patient iPSC-derived neurons (A–D′) High-resolution immunocytochemistry analyses of control TECPR2+/+ iPSC neurons (DIV45) treated with 5 μM scrambled non-targeting negative control ASO (scASO), single dose, gymnotic delivery at plating. Neurons were co-stained for MAP2 (A), β-III TUBULIN (B), and TECPR2 (D). β-III TUBULIN/TECPR2 merged signal (C), and an enlarged (∼2.1×) sub-region of the TECPR2 image (D′) are also presented. Distinct patterns of TECPR2 sub-cellular localization are highlighted in (D) and (D′): broad, speckled puncta pattern within soma and proximal dendrites (cyan arrowhead), small discrete puncta in neurites (yellow arrowhead), and rare large puncta in neurites (green arrowheads). (E–H′) Control TECPR2+/ - iPSC neurons (DIV45) treated with 5 μM scASO, single dose, gymnotic delivery show similar pattern of TECPR2 subcellular localization to that of TECPR2+/+ (D and D′) with the three types of puncta highlighted by arrowheads in (H) and (H′): broad puncta pattern within soma and proximal dendrites (cyan arrowhead), small discrete puncta in neurites (yellow arrowhead), and rare large puncta in neurites (green arrowheads). (I–L′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with the 5 μM scASO show no TECPR2 immunoreactivity and absence of the punctate expression pattern. White arrowhead in (L) and (L′) indicates lack of TECPR2 immunoreactivity signal in an area of a MAP2+ soma, according to (I). (M–P′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of broad puncta pattern of TECPR2 expression within soma and proximal dendrites, as highlighted by cyan arrowhead in (P) and enlarged (∼2.1×) in (P′). (Q–T′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of expression of small discrete TECPR2-positive puncta in β-III TUBULIN-positive neurites, as highlighted by yellow arrowheads in (T) and enlarged (∼2.1×) (T′). (U–X′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of expression of rare large TECPR2-positive puncta in neurites, as highlighted by green arrowhead in (X) and enlarged (∼2.1×) (X′). (A–X′) Scale bar, 20 μm.
Figure Legend Snippet: TECPR2 and TECPR2ΔExon8 neuronal localization in patient iPSC-derived neurons (A–D′) High-resolution immunocytochemistry analyses of control TECPR2+/+ iPSC neurons (DIV45) treated with 5 μM scrambled non-targeting negative control ASO (scASO), single dose, gymnotic delivery at plating. Neurons were co-stained for MAP2 (A), β-III TUBULIN (B), and TECPR2 (D). β-III TUBULIN/TECPR2 merged signal (C), and an enlarged (∼2.1×) sub-region of the TECPR2 image (D′) are also presented. Distinct patterns of TECPR2 sub-cellular localization are highlighted in (D) and (D′): broad, speckled puncta pattern within soma and proximal dendrites (cyan arrowhead), small discrete puncta in neurites (yellow arrowhead), and rare large puncta in neurites (green arrowheads). (E–H′) Control TECPR2+/ - iPSC neurons (DIV45) treated with 5 μM scASO, single dose, gymnotic delivery show similar pattern of TECPR2 subcellular localization to that of TECPR2+/+ (D and D′) with the three types of puncta highlighted by arrowheads in (H) and (H′): broad puncta pattern within soma and proximal dendrites (cyan arrowhead), small discrete puncta in neurites (yellow arrowhead), and rare large puncta in neurites (green arrowheads). (I–L′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with the 5 μM scASO show no TECPR2 immunoreactivity and absence of the punctate expression pattern. White arrowhead in (L) and (L′) indicates lack of TECPR2 immunoreactivity signal in an area of a MAP2+ soma, according to (I). (M–P′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of broad puncta pattern of TECPR2 expression within soma and proximal dendrites, as highlighted by cyan arrowhead in (P) and enlarged (∼2.1×) in (P′). (Q–T′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of expression of small discrete TECPR2-positive puncta in β-III TUBULIN-positive neurites, as highlighted by yellow arrowheads in (T) and enlarged (∼2.1×) (T′). (U–X′) SPG49 patient TECPR2-/- iPSC-derived NGN2 neurons (DIV45) treated with 5 μM ASO-005-02 (single dose, gymnotic delivery at plating) show rescue of expression of rare large TECPR2-positive puncta in neurites, as highlighted by green arrowhead in (X) and enlarged (∼2.1×) (X′). (A–X′) Scale bar, 20 μm.

Techniques Used: Derivative Assay, Immunocytochemistry, Negative Control, Allele-specific Oligonucleotide, Staining, Expressing

8) Product Images from "An epilepsy‐associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model. An epilepsy‐associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model"

Article Title: An epilepsy‐associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model. An epilepsy‐associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model

Journal: Journal of Neuroscience Research

doi: 10.1002/jnr.24747

Neural differentiation of patient‐derived induced pluripotent stem cells (iPSCs) to neural progenitor cells and neurons. (a) Schematic of neural differentiation from EIEE‐76 patient‐specific iPSCs. (b) Neural progenitor cells (NPCs) differentiated from control (Unaffected: CW067 U5; CW067 U9) and an EIEE‐76 patient (Affected: CW066 A4; CW066 A5) iPSC clones are indistinguishable. The iPSCs clones form embryoid bodies (EBs) and neural rosettes (yellow, dashed outline) similarly upon neural induction. Isolated NPCs express neural progenitor cell markers PAX6 and NESTIN. Representative images shown with DAPI (4′,6‐diamidino‐2‐phenylindole) counterstain. Scale bar, 100 μm. (c) Characterization of NPC lines derived from unaffected familial control (CW067) and an EIEE‐76 affected patient (CW066) by RT‐PCR for expression of neural crest cell ( SOX10 and FOXD3), stem cell ( OCT4), and neural progenitor cell ( NESTIN ) identity genes show the NPC cultures are not contaminated by neural crest derivatives or undifferentiated stem cells. GAPDH , loading control. (d) Co‐immunocytochemistry for neuronal markers MAP2 and TUJ1 at day (d)25 of differentiation show positive expression in familial unaffected controls and EIEE‐76 patient‐specific affected neurons. Representative images shown with DAPI counterstain. Scale bar, 50 μm. (e) Dual color infrared western blot for HA (green) and ACTL6B (red) in HEK293T cell lysates transfected with a 2xHA‐epitope tagged ACTL6B expression construct showing strong colocalization at the expected size of 49 kDa. (f) Temporal analysis of ACTL6B expression during neural differentiation found ACTL6B was detected at increasing levels after day d3, while TUJ1 expression was first visible at d10. GAPDH, loading control. Western blot from neurons differentiated from iPSC clone CW067 U9 is displayed
Figure Legend Snippet: Neural differentiation of patient‐derived induced pluripotent stem cells (iPSCs) to neural progenitor cells and neurons. (a) Schematic of neural differentiation from EIEE‐76 patient‐specific iPSCs. (b) Neural progenitor cells (NPCs) differentiated from control (Unaffected: CW067 U5; CW067 U9) and an EIEE‐76 patient (Affected: CW066 A4; CW066 A5) iPSC clones are indistinguishable. The iPSCs clones form embryoid bodies (EBs) and neural rosettes (yellow, dashed outline) similarly upon neural induction. Isolated NPCs express neural progenitor cell markers PAX6 and NESTIN. Representative images shown with DAPI (4′,6‐diamidino‐2‐phenylindole) counterstain. Scale bar, 100 μm. (c) Characterization of NPC lines derived from unaffected familial control (CW067) and an EIEE‐76 affected patient (CW066) by RT‐PCR for expression of neural crest cell ( SOX10 and FOXD3), stem cell ( OCT4), and neural progenitor cell ( NESTIN ) identity genes show the NPC cultures are not contaminated by neural crest derivatives or undifferentiated stem cells. GAPDH , loading control. (d) Co‐immunocytochemistry for neuronal markers MAP2 and TUJ1 at day (d)25 of differentiation show positive expression in familial unaffected controls and EIEE‐76 patient‐specific affected neurons. Representative images shown with DAPI counterstain. Scale bar, 50 μm. (e) Dual color infrared western blot for HA (green) and ACTL6B (red) in HEK293T cell lysates transfected with a 2xHA‐epitope tagged ACTL6B expression construct showing strong colocalization at the expected size of 49 kDa. (f) Temporal analysis of ACTL6B expression during neural differentiation found ACTL6B was detected at increasing levels after day d3, while TUJ1 expression was first visible at d10. GAPDH, loading control. Western blot from neurons differentiated from iPSC clone CW067 U9 is displayed

Techniques Used: Derivative Assay, Clone Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunocytochemistry, Western Blot, Transfection, Construct

9) Product Images from "Loss of coiled-coil protein Cep55 impairs abscission processes and results in p53-dependent apoptosis in developing cortex"

Article Title: Loss of coiled-coil protein Cep55 impairs abscission processes and results in p53-dependent apoptosis in developing cortex

Journal: bioRxiv

doi: 10.1101/2020.06.02.129346

Cep55 knockout cortex at E14.5 shows reduced and disorganized neuron and NSC layers. (A) Representative images of cortical sections from Cep55 +/− and −/− E14.5 embryos immunostained for neuron marker Tubb3 (gray) show cortical plate (cp) and axons in the intermediate zone (iz). In −/− images, the cp is disorganized the border between cp and iz is unclear. (B) Mean thicknesses of total cortex and neuron layer are significantly decreased in −/− brains. (C) The −/− cp/iz occupies proportionally less of cortical width than normal, while the vz/svz occupies more . (D) Cortical sections stained for NSC marker Pax6 (red) and basal progenitor (BP) marker Tbr2 (green) show NSCs in the vz of Cep55−/− brains are more disorganized, with some empty spaces (square), and some nuclei mislocalized basally above the svz (arrows). (E-F) NSCs (Pax6+) per cortical length are reduced in Cep55−/− and some mislocalized. BP numbers (Tbr2+) are not significantly changed. For B, total thickness, n = 7 +/− and 8 −/− brains. For B, cp/iz and vz/svz thickness, and for C,E,F, n= 4 +/− and 4 −/− brains. (G) Phospho-histone H3 (PH3) immunostaining is used to mark cells in mitosis. (H) Cep55−/− cortices show a normal number of mitotic cells (PH3+, magenta) at the apical membrane but an increased number of mitotic cells basally. (I) The mitotic index of NSCs (Pax6+) is normal, but of BPs (Tbr2+) is significantly increased in Cep55 −/− cortices. Dashed line in A,D,G = apical membrane. For H,I: n= 4 Cep55+/− and 4 −/− brains. Scale bar: (A,G): 20 μm. n.s.; not significant, * p
Figure Legend Snippet: Cep55 knockout cortex at E14.5 shows reduced and disorganized neuron and NSC layers. (A) Representative images of cortical sections from Cep55 +/− and −/− E14.5 embryos immunostained for neuron marker Tubb3 (gray) show cortical plate (cp) and axons in the intermediate zone (iz). In −/− images, the cp is disorganized the border between cp and iz is unclear. (B) Mean thicknesses of total cortex and neuron layer are significantly decreased in −/− brains. (C) The −/− cp/iz occupies proportionally less of cortical width than normal, while the vz/svz occupies more . (D) Cortical sections stained for NSC marker Pax6 (red) and basal progenitor (BP) marker Tbr2 (green) show NSCs in the vz of Cep55−/− brains are more disorganized, with some empty spaces (square), and some nuclei mislocalized basally above the svz (arrows). (E-F) NSCs (Pax6+) per cortical length are reduced in Cep55−/− and some mislocalized. BP numbers (Tbr2+) are not significantly changed. For B, total thickness, n = 7 +/− and 8 −/− brains. For B, cp/iz and vz/svz thickness, and for C,E,F, n= 4 +/− and 4 −/− brains. (G) Phospho-histone H3 (PH3) immunostaining is used to mark cells in mitosis. (H) Cep55−/− cortices show a normal number of mitotic cells (PH3+, magenta) at the apical membrane but an increased number of mitotic cells basally. (I) The mitotic index of NSCs (Pax6+) is normal, but of BPs (Tbr2+) is significantly increased in Cep55 −/− cortices. Dashed line in A,D,G = apical membrane. For H,I: n= 4 Cep55+/− and 4 −/− brains. Scale bar: (A,G): 20 μm. n.s.; not significant, * p

Techniques Used: Knock-Out, Marker, Staining, Immunostaining

p53 nuclear accumulation is increased in Cep55 knockout binucleate NSCs and neurons, but not in binucleate MEFs. (A-B) Cortical sections immunostained for p53 and Tubb3 show almost no p53+ cells in controls but many in Cep55−/− cortices. Arrows, paired nuclei with p53 expression. B. p53+ cell counts are increased in Cep55−/− cortices at E10.5, and at E14.5, mostly in the vz/svz than neuronal layers (C) Images of dissociated NSC cultures show p53+ binucleate NSCs from Cep55 −/− cortices. (D) Counts of cells with a p53 nuclear:cytoplasmic (N:C) ratio of > 2 are greatly increased in −/− cultures, both NSCs and neurons. (E-F) In Cep55 −/− NSC cultures, the mean N:C ratio of p53 intensity is about 1 in mononucleate NSCs or neurons, but significantly higher in binucleate progenitors and neurons. (F) In Cep55−/− cultures, over half of binucleate NSCs have a p53 N:C ratio > 2, compared to only 1% of mononucleate NSCs. Among binucleate neurons, ~ 20% have a p53 N:C ratio of > 2 versus only 2% of mononucleate neurons. (H) Apoptosis (CC3+) is not increased in Cep55−/− primary MEF cultures compared to controls. (I) The N:C ratio of p53 signal (p53, green, G) is not different in −/− MEFs compared to controls. (J) Binucleate Cep55 −/− MEFs did not have an increased p53 N:C ratio compared to mononucleate −/− MEFs (G, arrows). Dashed line in A = apical membrane For B, n = 3 Cep55+/+;+/− and 3 Cep55 −/− mice at each age. For D-F, n= 4 Cep55+/+;+/− and 4 Cep55−/− coverslips from 2 embryos each; 124 control and 137 −/− NSCs; 143 −/− and 184 −/− neurons for N:C ratios. For H, n= 3 control and 3 Cep55−/− coverslips from 3 embryos each. For I, n= 195 control and 232 −/− cells from 3 control and Cep55−/− coverslips and embryos each. For J, n= 153 mononucleate and 25 binucleate control cells and 158 mononucleate and 68 binucleate −/− cells from 3 coverslips and 3 embryos each. Scale bars in A, C, G represent 20 μm. * p
Figure Legend Snippet: p53 nuclear accumulation is increased in Cep55 knockout binucleate NSCs and neurons, but not in binucleate MEFs. (A-B) Cortical sections immunostained for p53 and Tubb3 show almost no p53+ cells in controls but many in Cep55−/− cortices. Arrows, paired nuclei with p53 expression. B. p53+ cell counts are increased in Cep55−/− cortices at E10.5, and at E14.5, mostly in the vz/svz than neuronal layers (C) Images of dissociated NSC cultures show p53+ binucleate NSCs from Cep55 −/− cortices. (D) Counts of cells with a p53 nuclear:cytoplasmic (N:C) ratio of > 2 are greatly increased in −/− cultures, both NSCs and neurons. (E-F) In Cep55 −/− NSC cultures, the mean N:C ratio of p53 intensity is about 1 in mononucleate NSCs or neurons, but significantly higher in binucleate progenitors and neurons. (F) In Cep55−/− cultures, over half of binucleate NSCs have a p53 N:C ratio > 2, compared to only 1% of mononucleate NSCs. Among binucleate neurons, ~ 20% have a p53 N:C ratio of > 2 versus only 2% of mononucleate neurons. (H) Apoptosis (CC3+) is not increased in Cep55−/− primary MEF cultures compared to controls. (I) The N:C ratio of p53 signal (p53, green, G) is not different in −/− MEFs compared to controls. (J) Binucleate Cep55 −/− MEFs did not have an increased p53 N:C ratio compared to mononucleate −/− MEFs (G, arrows). Dashed line in A = apical membrane For B, n = 3 Cep55+/+;+/− and 3 Cep55 −/− mice at each age. For D-F, n= 4 Cep55+/+;+/− and 4 Cep55−/− coverslips from 2 embryos each; 124 control and 137 −/− NSCs; 143 −/− and 184 −/− neurons for N:C ratios. For H, n= 3 control and 3 Cep55−/− coverslips from 3 embryos each. For I, n= 195 control and 232 −/− cells from 3 control and Cep55−/− coverslips and embryos each. For J, n= 153 mononucleate and 25 binucleate control cells and 158 mononucleate and 68 binucleate −/− cells from 3 coverslips and 3 embryos each. Scale bars in A, C, G represent 20 μm. * p

Techniques Used: Knock-Out, Expressing, Mouse Assay

10) Product Images from "Angiomotin links ROCK and YAP signaling in mechanosensitive differentiation of neural stem cells"

Article Title: Angiomotin links ROCK and YAP signaling in mechanosensitive differentiation of neural stem cells

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E19-11-0602

AMOT promotes neurogenesis on soft substrates. (a) Western blot showing protein depletion and gene KO of AMOT in NSCs using shRNA and CRISPR/Cas9. (b) Representative immunofluorescence images of naive, AMOT KD (shAMOT), and AMOT KO (sgAMOT) NSCs after culture in mixed differentiation conditions (1 µM retinoic acid + 1% FBS) on soft (0.2 kPa) or stiff (73 kPa) substrates. Cells were fixed and stained for DAPI (magenta) and Tuj1 (green), a neuronal marker. Bar = 100 µm. (c) Quantification of neurogenesis was measured by the percentage of Tuj1+ cells after 6 d of differentiation. Error bars represent SD ( n = 3 gels). **** p
Figure Legend Snippet: AMOT promotes neurogenesis on soft substrates. (a) Western blot showing protein depletion and gene KO of AMOT in NSCs using shRNA and CRISPR/Cas9. (b) Representative immunofluorescence images of naive, AMOT KD (shAMOT), and AMOT KO (sgAMOT) NSCs after culture in mixed differentiation conditions (1 µM retinoic acid + 1% FBS) on soft (0.2 kPa) or stiff (73 kPa) substrates. Cells were fixed and stained for DAPI (magenta) and Tuj1 (green), a neuronal marker. Bar = 100 µm. (c) Quantification of neurogenesis was measured by the percentage of Tuj1+ cells after 6 d of differentiation. Error bars represent SD ( n = 3 gels). **** p

Techniques Used: Western Blot, shRNA, CRISPR, Immunofluorescence, Staining, Marker

AMOT phosphorylation and actin-binding regulate its neurogenic effect. (a) Schematic drawings depicting protein sequence of AMOT variants that were cloned into retroviral plasmids before being packaged and used to generate stable AMOT overexpression NSC cell lines. (b) Quantification of neurogenesis as the percentage of Tuj1+ cells after 6 d of differentiation. Error bars represent SD ( n = 3 gels). (c) Fluorescent immunohistochemical staining of a mouse hippocampal section. Tissue section was fixed and stained for DAPI (blue), DCX (green), an immature neuronal marker, and pAMOT (red). White arrowheads indicate DCX+/pAMOT+ cells. (d) Left: representative immunofluorescence images of WT, ΔAB, S175E, and S175A AMOT overexpression NSCs after 24 h of differentiation on stiff substrates. Myc antibody detects a C-terminal epitope tag only present on exogenous AMOT. Right: plotted intensity line traces from the Myc/AMOT and F-actin channels correlating to white dotted arrows shown on the left. Bars = 20 µm. ** p
Figure Legend Snippet: AMOT phosphorylation and actin-binding regulate its neurogenic effect. (a) Schematic drawings depicting protein sequence of AMOT variants that were cloned into retroviral plasmids before being packaged and used to generate stable AMOT overexpression NSC cell lines. (b) Quantification of neurogenesis as the percentage of Tuj1+ cells after 6 d of differentiation. Error bars represent SD ( n = 3 gels). (c) Fluorescent immunohistochemical staining of a mouse hippocampal section. Tissue section was fixed and stained for DAPI (blue), DCX (green), an immature neuronal marker, and pAMOT (red). White arrowheads indicate DCX+/pAMOT+ cells. (d) Left: representative immunofluorescence images of WT, ΔAB, S175E, and S175A AMOT overexpression NSCs after 24 h of differentiation on stiff substrates. Myc antibody detects a C-terminal epitope tag only present on exogenous AMOT. Right: plotted intensity line traces from the Myc/AMOT and F-actin channels correlating to white dotted arrows shown on the left. Bars = 20 µm. ** p

Techniques Used: Binding Assay, Sequencing, Clone Assay, Over Expression, Immunohistochemistry, Staining, Marker, Immunofluorescence

11) Product Images from "Sox11 Expression Promotes Regeneration of Some Retinal Ganglion Cell Types but Kills Others"

Article Title: Sox11 Expression Promotes Regeneration of Some Retinal Ganglion Cell Types but Kills Others

Journal: Neuron

doi: 10.1016/j.neuron.2017.05.035

Pro-regenerative effect of Sox11 in non-alpha-RGCs is enhanced by PTEN deletion (A) Representative images of optic nerves showing regenerating axons from different groups at 6–7 weeks after injury. (B) Quantification of regenerating axons shown in (A). (C) Images showing regenerating axons in ΔPTEN/Sox11-treated mice in the optic tract. (D) RGC survival results at 6–7 weeks after injury. (E) Retinal sections from ΔPTEN/Sox11-treated mice with FluoroGold (FG) injection to the distal optic nerve, and stained with RBPMS or TUJ1 antibodies. (F) Quantification showing percentages of RBPMS- and TUJ1-co-stained cells among FG-positive cells shown in (E). (G, H) Retinal images (G) and quantification (H) showing co-staining of FG-labeled RGCs with anti-OPN or SMI32. In ΔPTEN-treated animals, 80% of FG-labeled cells are both OPN-positive and SMI32-positive; however, none of the FG-labeled cells are co-stained with either OPN or SMI32 in ΔPTEN/Sox11-treated animals. Scale bar in (A) represents 500 µm. Scale bars in (C), (E) and (G) represent 100 µm. Data in (B) are expressed as mean ± SEM while data in (D), (F) and (H) are expressed as mean ± SD (n = 3–5). Significance levels in (B) are indicated by *, P
Figure Legend Snippet: Pro-regenerative effect of Sox11 in non-alpha-RGCs is enhanced by PTEN deletion (A) Representative images of optic nerves showing regenerating axons from different groups at 6–7 weeks after injury. (B) Quantification of regenerating axons shown in (A). (C) Images showing regenerating axons in ΔPTEN/Sox11-treated mice in the optic tract. (D) RGC survival results at 6–7 weeks after injury. (E) Retinal sections from ΔPTEN/Sox11-treated mice with FluoroGold (FG) injection to the distal optic nerve, and stained with RBPMS or TUJ1 antibodies. (F) Quantification showing percentages of RBPMS- and TUJ1-co-stained cells among FG-positive cells shown in (E). (G, H) Retinal images (G) and quantification (H) showing co-staining of FG-labeled RGCs with anti-OPN or SMI32. In ΔPTEN-treated animals, 80% of FG-labeled cells are both OPN-positive and SMI32-positive; however, none of the FG-labeled cells are co-stained with either OPN or SMI32 in ΔPTEN/Sox11-treated animals. Scale bar in (A) represents 500 µm. Scale bars in (C), (E) and (G) represent 100 µm. Data in (B) are expressed as mean ± SEM while data in (D), (F) and (H) are expressed as mean ± SD (n = 3–5). Significance levels in (B) are indicated by *, P

Techniques Used: Mouse Assay, Injection, Staining, Labeling

12) Product Images from "Pharmacotherapy alleviates pathological changes in human direct reprogrammed neuronal cell model of myotonic dystrophy type 1"

Article Title: Pharmacotherapy alleviates pathological changes in human direct reprogrammed neuronal cell model of myotonic dystrophy type 1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0269683

Direct reprogramming of DM1 patients’ skin fibroblasts into neuronal like cells. (a) Differentiation process of DM1 skin fibroblasts into neuronal like cells. The top left image shows DM1 skin fibroblasts before adding lentivirus co-expressing shRNA against PTBP and puromycin resistant gene. The top middle image shows lentivirus transduced cells successfully passing the selection process by adding 5 μg/mL puromycin to the culture medium for 2 days. The top right image shows expansion of cell bodies at 6 DPI. The bottom left image shows cells with neuronal like morphology of expanded cell bodies and distinct neurites at 8 DPI. The bottom right image shows bipolar and multipolar neurons with extended neurites at 10 DPI. Scale bar, 50 μm. (b) Co-immunostaining of control (top panel) and DM1 (bottom panel) hiNeurons with a neuron specific β-III tubulin antibody (TUJ1) (green) and MAP2 (red) at 10 DPI. DAPI was used to stain nuclei (blue). On the right: merged images of immunostaining with TUJ1, MAP2 and DAPI. Scale bar, 50 μm. (c) Graph shows the percentage of positive TUJ1-stained cells (TUJ1 + ) at 10 DPI, quantified by dividing the number of TUJ1 + cells by the total number of nuclei stained by DAPI. Data are shown as mean ± SEM; n = 3 for each group, a total of 90 images were analyzed per group (11,510 nuclei were analyzed per ctrl group and 14,668 nuclei per DM1 group). ns, not significant compared to control group by unpaired t-test. (d) Graph displays the percentage of MAP2 positive cells at 10 DPI, quantified by dividing the number of MAP2 + cells by the total number of TUJ1 + cells. Data are shown as mean ± SEM; n = 3 for each group, a total of 90 images were analyzed per group (9,941 TUJ1 + cells were analyzed per ctrl group and 16,286 TUJ1 + cells per DM1 group). ns, not significant compared to control group by unpaired t-test. (e) Additional staining for neural markers. Fluorescent images display positive co-staining of DM1 hiNeurons with TUJ1 (green) and anti GABA or glutamate or NeuN (red) antibodies at 15 DPI. The bottom panel shows lack of synapse formation at 10 DPI in DM1 hiNeurons co-stained with TUJ1 (green) and SYN1 (red) antibodies. DAPI was used to stain nuclei (blue). On the right merged images. Scale bar, 50 μm. Ctrl, control; DM1, myotonic dystrophy type 1; hiN, human induced neurons; DPI, days post viral-infection; TUJ1, β-III tubulin antibody; MAP2, microtubule-associated protein 2; SEM, standard error of the mean; GABA, gamma-aminobutyric acid; NeuN, neuronal nuclear antibody; SYN1, synapsin 1 antibody.
Figure Legend Snippet: Direct reprogramming of DM1 patients’ skin fibroblasts into neuronal like cells. (a) Differentiation process of DM1 skin fibroblasts into neuronal like cells. The top left image shows DM1 skin fibroblasts before adding lentivirus co-expressing shRNA against PTBP and puromycin resistant gene. The top middle image shows lentivirus transduced cells successfully passing the selection process by adding 5 μg/mL puromycin to the culture medium for 2 days. The top right image shows expansion of cell bodies at 6 DPI. The bottom left image shows cells with neuronal like morphology of expanded cell bodies and distinct neurites at 8 DPI. The bottom right image shows bipolar and multipolar neurons with extended neurites at 10 DPI. Scale bar, 50 μm. (b) Co-immunostaining of control (top panel) and DM1 (bottom panel) hiNeurons with a neuron specific β-III tubulin antibody (TUJ1) (green) and MAP2 (red) at 10 DPI. DAPI was used to stain nuclei (blue). On the right: merged images of immunostaining with TUJ1, MAP2 and DAPI. Scale bar, 50 μm. (c) Graph shows the percentage of positive TUJ1-stained cells (TUJ1 + ) at 10 DPI, quantified by dividing the number of TUJ1 + cells by the total number of nuclei stained by DAPI. Data are shown as mean ± SEM; n = 3 for each group, a total of 90 images were analyzed per group (11,510 nuclei were analyzed per ctrl group and 14,668 nuclei per DM1 group). ns, not significant compared to control group by unpaired t-test. (d) Graph displays the percentage of MAP2 positive cells at 10 DPI, quantified by dividing the number of MAP2 + cells by the total number of TUJ1 + cells. Data are shown as mean ± SEM; n = 3 for each group, a total of 90 images were analyzed per group (9,941 TUJ1 + cells were analyzed per ctrl group and 16,286 TUJ1 + cells per DM1 group). ns, not significant compared to control group by unpaired t-test. (e) Additional staining for neural markers. Fluorescent images display positive co-staining of DM1 hiNeurons with TUJ1 (green) and anti GABA or glutamate or NeuN (red) antibodies at 15 DPI. The bottom panel shows lack of synapse formation at 10 DPI in DM1 hiNeurons co-stained with TUJ1 (green) and SYN1 (red) antibodies. DAPI was used to stain nuclei (blue). On the right merged images. Scale bar, 50 μm. Ctrl, control; DM1, myotonic dystrophy type 1; hiN, human induced neurons; DPI, days post viral-infection; TUJ1, β-III tubulin antibody; MAP2, microtubule-associated protein 2; SEM, standard error of the mean; GABA, gamma-aminobutyric acid; NeuN, neuronal nuclear antibody; SYN1, synapsin 1 antibody.

Techniques Used: Expressing, shRNA, Selection, Immunostaining, Staining, Infection

Abnormal axonal outgrowth in DM1 hiNeurons at 15 DPI. (a) Representative fluorescent images display axons of control (top panel) and DM1 (bottom panel) hiNeurons visualized by co-staining with TUJ1 (green) and SMI-312 (red) antibodies. Nuclei stained by DAPI. On the right merged images of TUJ1, SMI-312 and DAPI. Scale bar, 50 μm. (b-e) box and whisker plots show measurements of axonal length of control and DM1 hiNeurons. Results were categorized into four categories: the longest/photo (b), medium length/photo (c), the shortest/photo (d) and average/photo (e). Each sample is shown in different color. Line (+) sign inside the box represent median and mean of replicates (outcome analyzed), respectively. Whiskers show minimum maximum values. Note that the average length of axons measured in DM1 hiNeurons group is about half length of axons in control group. n = 3 for each group, a total of 36 (the longest, medium and the shortest/photo analysis) or 108 (average/photo analysis) axons were analyzed per sample. A total of 36 images were analyzed per sample. **P
Figure Legend Snippet: Abnormal axonal outgrowth in DM1 hiNeurons at 15 DPI. (a) Representative fluorescent images display axons of control (top panel) and DM1 (bottom panel) hiNeurons visualized by co-staining with TUJ1 (green) and SMI-312 (red) antibodies. Nuclei stained by DAPI. On the right merged images of TUJ1, SMI-312 and DAPI. Scale bar, 50 μm. (b-e) box and whisker plots show measurements of axonal length of control and DM1 hiNeurons. Results were categorized into four categories: the longest/photo (b), medium length/photo (c), the shortest/photo (d) and average/photo (e). Each sample is shown in different color. Line (+) sign inside the box represent median and mean of replicates (outcome analyzed), respectively. Whiskers show minimum maximum values. Note that the average length of axons measured in DM1 hiNeurons group is about half length of axons in control group. n = 3 for each group, a total of 36 (the longest, medium and the shortest/photo analysis) or 108 (average/photo analysis) axons were analyzed per sample. A total of 36 images were analyzed per sample. **P

Techniques Used: Staining, Whisker Assay

Accumulation of nuclear RNA foci in DM1 hiNeurons at 10 DPI and their reduction by ACT treatment. (a) FISH with 5’ Texas red 2-O-methyl- CAG RNA probe and immunostaining with TUJ1 and DAPI revealed punctate and discrete nuclear RNA foci (red) in DM1 hiNeurons (a2) but not in control hiNeurons (a1). Nuclear RNA foci were abundant in DM1 hiNeurons (a2) but treatment with 100 nM (a3) or 200 nM (a4) ACT reduced them remarkably (left: FISH, middle: merged images of FISH and DAPI staining nuclei (blue), right: merged images of FISH, DAPI and TUJ1 antibody staining neurons (green)). Scale bar, 20 μm. (b) Scatter plot shows percentage of nuclei containing RNA foci in DM1 hiNeurons. Each sample is presented in different color. Each symbol represents the percentage of nuclei containing RNA foci for each sample replicate. Line represents the mean. Note that nuclear RNA foci were present in most of DM1 hiNeurons. (c) Box and whisker plot shows number of RNA foci per nucleus in DM1 hiNeurons. Each sample is presented in different color. Line and (+) sign inside the box represent median and mean of replicates (outcome analyzed), respectively. Whiskers show minimum maximum values. Counting was performed manually. n = 3 for each group, a total of 324 nuclei were analyzed per sample. ****P
Figure Legend Snippet: Accumulation of nuclear RNA foci in DM1 hiNeurons at 10 DPI and their reduction by ACT treatment. (a) FISH with 5’ Texas red 2-O-methyl- CAG RNA probe and immunostaining with TUJ1 and DAPI revealed punctate and discrete nuclear RNA foci (red) in DM1 hiNeurons (a2) but not in control hiNeurons (a1). Nuclear RNA foci were abundant in DM1 hiNeurons (a2) but treatment with 100 nM (a3) or 200 nM (a4) ACT reduced them remarkably (left: FISH, middle: merged images of FISH and DAPI staining nuclei (blue), right: merged images of FISH, DAPI and TUJ1 antibody staining neurons (green)). Scale bar, 20 μm. (b) Scatter plot shows percentage of nuclei containing RNA foci in DM1 hiNeurons. Each sample is presented in different color. Each symbol represents the percentage of nuclei containing RNA foci for each sample replicate. Line represents the mean. Note that nuclear RNA foci were present in most of DM1 hiNeurons. (c) Box and whisker plot shows number of RNA foci per nucleus in DM1 hiNeurons. Each sample is presented in different color. Line and (+) sign inside the box represent median and mean of replicates (outcome analyzed), respectively. Whiskers show minimum maximum values. Counting was performed manually. n = 3 for each group, a total of 324 nuclei were analyzed per sample. ****P

Techniques Used: Fluorescence In Situ Hybridization, Immunostaining, Staining, Whisker Assay

13) Product Images from "GSK3β negatively regulates TRAX, a scaffold protein implicated in mental disorders, for NHEJ-mediated DNA repair in neurons"

Article Title: GSK3β negatively regulates TRAX, a scaffold protein implicated in mental disorders, for NHEJ-mediated DNA repair in neurons

Journal: Molecular Psychiatry

doi: 10.1038/s41380-017-0007-z

The protective effect of an A 2A R agonist is mediated by TRAX. a-c PC12-pSuper and PC12-shTRAX cells were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-TRAX and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times ( a ). The extent of DNA damage was analyzed via the neutral comet assay. The mean tail moment was quantified with COMETscore.v1.5 software, scale bar, 50 μm ( b ). Apoptosis was assessed with the Annexin V apoptosis detection kit. Cells were co-stained with Annexin V-FITC and PI for 10 min followed by flow cytometric analysis. These experiments were repeated three times ( c ). d , e Primary hippocampal neurons (DIV14) from TRAX-WT and TRAX-null mice were treated with CGS (10 μM) or vehicle for 1 h and then treated with H 2 O 2 (100 μM) for 2 h. DNA damage was assessed by determining the number of DNA foci per cell by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). The percentage of cells with > 5 γH2AX foci per cell in at least 100 cells were determined in each condition. Scale bar, 10 μm. f Human MSN neurons were infected with lentivirus expressing TRAX shRNA or control shRNA for 3 days. Human MSN neurons were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-TRAX and anti-β-Actin antibodies as indicated. The amount of target protein was quantified and normalized to that of β-Actin, the loading control. These experiments were repeated three times. Data are presented as the mean ± SEM from at least three independent experiments. * P
Figure Legend Snippet: The protective effect of an A 2A R agonist is mediated by TRAX. a-c PC12-pSuper and PC12-shTRAX cells were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-TRAX and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times ( a ). The extent of DNA damage was analyzed via the neutral comet assay. The mean tail moment was quantified with COMETscore.v1.5 software, scale bar, 50 μm ( b ). Apoptosis was assessed with the Annexin V apoptosis detection kit. Cells were co-stained with Annexin V-FITC and PI for 10 min followed by flow cytometric analysis. These experiments were repeated three times ( c ). d , e Primary hippocampal neurons (DIV14) from TRAX-WT and TRAX-null mice were treated with CGS (10 μM) or vehicle for 1 h and then treated with H 2 O 2 (100 μM) for 2 h. DNA damage was assessed by determining the number of DNA foci per cell by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). The percentage of cells with > 5 γH2AX foci per cell in at least 100 cells were determined in each condition. Scale bar, 10 μm. f Human MSN neurons were infected with lentivirus expressing TRAX shRNA or control shRNA for 3 days. Human MSN neurons were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-TRAX and anti-β-Actin antibodies as indicated. The amount of target protein was quantified and normalized to that of β-Actin, the loading control. These experiments were repeated three times. Data are presented as the mean ± SEM from at least three independent experiments. * P

Techniques Used: SDS Page, Western Blot, Neutral Comet Assay, Software, Staining, Flow Cytometry, Mouse Assay, Immunofluorescence, Marker, Infection, Expressing, shRNA

Activation of the A 2A R ameliorates oxidative stress-induced DNA damage and toxicity. a PC12 cells were treated with an agonist of the A 2A R (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R, followed by the addition of H 2 O 2 (100 μM) for 4 h. The extent of DNA damage was analyzed via the neutral comet assay. The mean tail moment was quantified by using COMETscore.v1.5 software. Scale bar, 50 μm. b PC12 cells were pretreated with CGS (10 μM) or vehicle for 1 h and then treated with H 2 O 2 (100 μM) for the indicated time. Cells were lysed and subjected to SDS–PAGE and Western blot analysis using the anti-γH2AX and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c Primary hippocampal neurons (DIV14) harvested from wild-type B6/C57 mice were treated with CGS (10 μM) or vehicle for 1 h and then treated with H 2 O 2 (100 μM) for 2 h. DNA damage was assessed by determining the number of DNA foci per cell by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). The percentage of cells with > 5 γH2AX foci per cell in at least 100 cells were determined in each condition. Scale bar, 10 μm. d PC12 cells were treated with CGS (10 μM) or vehicle for 1 h, followed by treatment with H 2 O 2 (100 μM) for 4 h. The survival of these treated cells was measured using the Annexin V apoptosis detection assay kit. Cells were co-stained with Annexin V-FITC and propidium iodide (PI) for 10 min followed by flow cytometric analysis. These experiments were repeated three times. e , f PC12 cells were treated with an inhibitor of protein kinase A (H89; 10 μM), an A 2A R-selective inhibitor (SCH58261, SCH; 1 μM) or vehicle for 30 min and then treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R, followed by the addition of H 2 O 2 (100 μM) for 4 h ( e ). A123, a cAMP-dependent protein kinase (PKA)-deficient PC12 cell line, was treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R, followed by treatment with H 2 O 2 (100 μM) for 4 h ( f ). Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. Data are presented as the mean ± SEM from at least three independent experiments. ** P
Figure Legend Snippet: Activation of the A 2A R ameliorates oxidative stress-induced DNA damage and toxicity. a PC12 cells were treated with an agonist of the A 2A R (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R, followed by the addition of H 2 O 2 (100 μM) for 4 h. The extent of DNA damage was analyzed via the neutral comet assay. The mean tail moment was quantified by using COMETscore.v1.5 software. Scale bar, 50 μm. b PC12 cells were pretreated with CGS (10 μM) or vehicle for 1 h and then treated with H 2 O 2 (100 μM) for the indicated time. Cells were lysed and subjected to SDS–PAGE and Western blot analysis using the anti-γH2AX and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c Primary hippocampal neurons (DIV14) harvested from wild-type B6/C57 mice were treated with CGS (10 μM) or vehicle for 1 h and then treated with H 2 O 2 (100 μM) for 2 h. DNA damage was assessed by determining the number of DNA foci per cell by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). The percentage of cells with > 5 γH2AX foci per cell in at least 100 cells were determined in each condition. Scale bar, 10 μm. d PC12 cells were treated with CGS (10 μM) or vehicle for 1 h, followed by treatment with H 2 O 2 (100 μM) for 4 h. The survival of these treated cells was measured using the Annexin V apoptosis detection assay kit. Cells were co-stained with Annexin V-FITC and propidium iodide (PI) for 10 min followed by flow cytometric analysis. These experiments were repeated three times. e , f PC12 cells were treated with an inhibitor of protein kinase A (H89; 10 μM), an A 2A R-selective inhibitor (SCH58261, SCH; 1 μM) or vehicle for 30 min and then treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R, followed by the addition of H 2 O 2 (100 μM) for 4 h ( e ). A123, a cAMP-dependent protein kinase (PKA)-deficient PC12 cell line, was treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R, followed by treatment with H 2 O 2 (100 μM) for 4 h ( f ). Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. Data are presented as the mean ± SEM from at least three independent experiments. ** P

Techniques Used: Activation Assay, Neutral Comet Assay, Software, SDS Page, Western Blot, Mouse Assay, Immunofluorescence, Staining, Marker, Detection Assay, Flow Cytometry

Inhibition of GSK3β enables TRAX-dependent DNA repair. a , b PC12-pSuper and PC12-shTRAX cells were treated with a GSK3β inhibitor (SB216763, 10 μM) or vehicle for 2 days. After the abovementioned treatment, cells were subjected to H 2 O 2 (100 μM) for 1–4 h as indicated. The levels of γH2AX, β-catenin, and α-Tubulin (a loading control) were evaluated by Western blot analysis using the indicated antibodies. The relative amounts of target proteins were quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c Primary hippocampal neurons (DIV14) harvested from wild-type B6/C57 mice were treated with SB216763 (10 μM) or vehicle for 1 day and then treated with H 2 O 2 (100 μM) for 2 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX and anti-α-Tubulin antibodies, as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. d Human iPSCs-derived MSN neurons were treated with SB216763 (10 μM) or vehicle for 1 day and then treated with H 2 O 2 (100 μM) for 4 h. DNA damage was assessed by determining the intensity of the DNA damage marker γH2AX by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). Scale bar, 50 μm. e PC12 cells were treated with a GSK3β inhibitor (SB216763, 10 μM) or vehicle for 2 days and nuclear and cytosolic fractions were isolated. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-TRAX, anti-α-Tubulin and anti-PARP antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin or PARP, the loading controls for post-nucleus and nucleus fractions, respectively. These experiments were repeated three times. The data are presented as the mean ± SEM from at least three independent experiments0. * P
Figure Legend Snippet: Inhibition of GSK3β enables TRAX-dependent DNA repair. a , b PC12-pSuper and PC12-shTRAX cells were treated with a GSK3β inhibitor (SB216763, 10 μM) or vehicle for 2 days. After the abovementioned treatment, cells were subjected to H 2 O 2 (100 μM) for 1–4 h as indicated. The levels of γH2AX, β-catenin, and α-Tubulin (a loading control) were evaluated by Western blot analysis using the indicated antibodies. The relative amounts of target proteins were quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c Primary hippocampal neurons (DIV14) harvested from wild-type B6/C57 mice were treated with SB216763 (10 μM) or vehicle for 1 day and then treated with H 2 O 2 (100 μM) for 2 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX and anti-α-Tubulin antibodies, as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. d Human iPSCs-derived MSN neurons were treated with SB216763 (10 μM) or vehicle for 1 day and then treated with H 2 O 2 (100 μM) for 4 h. DNA damage was assessed by determining the intensity of the DNA damage marker γH2AX by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). Scale bar, 50 μm. e PC12 cells were treated with a GSK3β inhibitor (SB216763, 10 μM) or vehicle for 2 days and nuclear and cytosolic fractions were isolated. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-TRAX, anti-α-Tubulin and anti-PARP antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin or PARP, the loading controls for post-nucleus and nucleus fractions, respectively. These experiments were repeated three times. The data are presented as the mean ± SEM from at least three independent experiments0. * P

Techniques Used: Inhibition, Western Blot, Mouse Assay, SDS Page, Derivative Assay, Marker, Immunofluorescence, Staining, Isolation

14) Product Images from "Target-agnostic discovery of Rett Syndrome therapeutics by coupling computational network analysis and CRISPR-enabled in vivo disease modeling"

Article Title: Target-agnostic discovery of Rett Syndrome therapeutics by coupling computational network analysis and CRISPR-enabled in vivo disease modeling

Journal: bioRxiv

doi: 10.1101/2022.03.20.485056

Network-based computational prediction of effective drugs to treat Rett syndrome in tadpole models. a, Network model for causality-aware discovery (nemoCAD) combines a directed gene-gene and drug-gene interaction network extracted from CTD, TRRUST, and KEGG databases with interaction probabilities inferred from single gene and drug perturbations in LINCS. Transcriptome data from any disease model or patient and corresponding control are used to identify the relevant subnetwork and disease-specific “node weights” that account for probabilities of up-/down-regulation of a gene. A drug-gene interaction probability matrix, inferred from LINCS, is computationally screened against the disease-specific subnetwork to identify compounds that significantly interact with the subnetwork and are ranked by their predicted ability to restore the disease transcriptome back to a healthy state based on single gene and gene network signatures. Downstream analyses can be performed on the resulting gene-gene interaction subnetwork by interrogating the underlying subnetwork structure to find control nodes and other network metrics. Additionally, the chemical structures of the predicted drugs can be clustered by structural similarity based on SMILES notation and annotated protein targets and pathways from DrugBank data. b , Seizure scores for wildtype tadpoles with vehicle alone (WT), MeCP2 knockdown tadpoles (MeCP2 KD), and MeCP2 KD animals treated with indicated doses of trofinetide (Tro), vorinostat (Vor), or ivermectin (Ive) for 0 to 8 days duration starting one week post-fertilization (scale bar at right indicates mean seizure score). c , Graphs showing relative effects on seizure score over 10 days of treatment in MeCP2 KD tadpoles of vehicle and 25 μM vorinostat (Vor) versus 70 μg/mL trofinetide (Tro), a clinical-stage drug with demonstrated efficacy (tadpoles per condition and timepoint: N = 12 Vor and Vehicle, N = 8 Tro; error bars indicate s.d.; ANOVA P = 0.028 Vehicle-treated MeCP2 KD tadpoles did not survive past day 3 of the treatment period in one study and past day 6 in a second study. Immunofluorescence micrographs ( d ) and graphs quantifying cilia orientation ( e ) and length ( f ) showing that abnormalities due to MeCP2 knockdown were restored by treatment with vorinostat (magenta-yellow colormap of fluorescence intensity, cilia stained for tubulin; ****, P
Figure Legend Snippet: Network-based computational prediction of effective drugs to treat Rett syndrome in tadpole models. a, Network model for causality-aware discovery (nemoCAD) combines a directed gene-gene and drug-gene interaction network extracted from CTD, TRRUST, and KEGG databases with interaction probabilities inferred from single gene and drug perturbations in LINCS. Transcriptome data from any disease model or patient and corresponding control are used to identify the relevant subnetwork and disease-specific “node weights” that account for probabilities of up-/down-regulation of a gene. A drug-gene interaction probability matrix, inferred from LINCS, is computationally screened against the disease-specific subnetwork to identify compounds that significantly interact with the subnetwork and are ranked by their predicted ability to restore the disease transcriptome back to a healthy state based on single gene and gene network signatures. Downstream analyses can be performed on the resulting gene-gene interaction subnetwork by interrogating the underlying subnetwork structure to find control nodes and other network metrics. Additionally, the chemical structures of the predicted drugs can be clustered by structural similarity based on SMILES notation and annotated protein targets and pathways from DrugBank data. b , Seizure scores for wildtype tadpoles with vehicle alone (WT), MeCP2 knockdown tadpoles (MeCP2 KD), and MeCP2 KD animals treated with indicated doses of trofinetide (Tro), vorinostat (Vor), or ivermectin (Ive) for 0 to 8 days duration starting one week post-fertilization (scale bar at right indicates mean seizure score). c , Graphs showing relative effects on seizure score over 10 days of treatment in MeCP2 KD tadpoles of vehicle and 25 μM vorinostat (Vor) versus 70 μg/mL trofinetide (Tro), a clinical-stage drug with demonstrated efficacy (tadpoles per condition and timepoint: N = 12 Vor and Vehicle, N = 8 Tro; error bars indicate s.d.; ANOVA P = 0.028 Vehicle-treated MeCP2 KD tadpoles did not survive past day 3 of the treatment period in one study and past day 6 in a second study. Immunofluorescence micrographs ( d ) and graphs quantifying cilia orientation ( e ) and length ( f ) showing that abnormalities due to MeCP2 knockdown were restored by treatment with vorinostat (magenta-yellow colormap of fluorescence intensity, cilia stained for tubulin; ****, P

Techniques Used: Immunofluorescence, Fluorescence, Staining

15) Product Images from "Exploration of Sensory and Spinal Neurons Expressing GRP in Itch and Pain"

Article Title: Exploration of Sensory and Spinal Neurons Expressing GRP in Itch and Pain

Journal: bioRxiv

doi: 10.1101/472886

Opto-activation of Grp + sensory neuron skin fibers evokes itch behavior. ( A ) Schematic of Grp Cre KI mating with Ai32 ChR2-eYFP line to produce Grp ChR2 mice. ( B ) IHC Image of eYFP expression in Grp ChR2 DRG. Scale bar in B , 50 μm. ( C ) IHC images of eYFP and ßlll-Tubulin in Grp ChR2 nape skin. Dashed lines indicate epidermal/dermal boundary. Scale bar in C , 100 μm. ( D ) Optical parameters of skin fiber stimulation Grp ChR2 and Grp WT mice. ( E ) Raster plot of scratching behavior induced by light stimulation of skin in Grp ChR2 and Grp WT mice. ( F ) Snapshots of Grp ChR2 and Grp WT mice with light off or on. Arrow indicates hind paw scratching the nape when light is on. ( G ) Total number of scratches during 5-min light stimulation experiment in Grp WT , Grp ChR2 , Grp ChR2 morphine-treated and Grp ChR2 BB-sap-treated mice. ( H ) n = 8 – 10 mice, one-way ANOVA with Tukey post hoc , *** p
Figure Legend Snippet: Opto-activation of Grp + sensory neuron skin fibers evokes itch behavior. ( A ) Schematic of Grp Cre KI mating with Ai32 ChR2-eYFP line to produce Grp ChR2 mice. ( B ) IHC Image of eYFP expression in Grp ChR2 DRG. Scale bar in B , 50 μm. ( C ) IHC images of eYFP and ßlll-Tubulin in Grp ChR2 nape skin. Dashed lines indicate epidermal/dermal boundary. Scale bar in C , 100 μm. ( D ) Optical parameters of skin fiber stimulation Grp ChR2 and Grp WT mice. ( E ) Raster plot of scratching behavior induced by light stimulation of skin in Grp ChR2 and Grp WT mice. ( F ) Snapshots of Grp ChR2 and Grp WT mice with light off or on. Arrow indicates hind paw scratching the nape when light is on. ( G ) Total number of scratches during 5-min light stimulation experiment in Grp WT , Grp ChR2 , Grp ChR2 morphine-treated and Grp ChR2 BB-sap-treated mice. ( H ) n = 8 – 10 mice, one-way ANOVA with Tukey post hoc , *** p

Techniques Used: Activation Assay, Mouse Assay, Immunohistochemistry, Expressing

( A ) IHC images of eYFP, CGRP and IB4 in Grp ChR2 DRG. Scale bar in A , 50 μm. ( B ) eYFP image from Grp WT ; Ai32 DRG. Scale bar in B , 50 μm. ( C ) IHC image of eYFP, βlII-Tubulin and DAPI in Grp WT ; Ai32 nape skin. Scale bar in C , 100 μm. ( D ) IHC image of eYFP and ßIII-Tubulin in Grp ChR2 cheek skin. Scale bar in D , 100 μm. ( E ) IHC images of eYFP, CGRP and DAPI merge in Grp ChR2 glabrous skin. Scale bar in E , 100 μm. ( F ) IHC image of eYFP and DiI in Grp ChR2 DRG or TG 10 days after i.d. nape or cheek injection of DiI tracer. Scale bar in F , 50 μm.
Figure Legend Snippet: ( A ) IHC images of eYFP, CGRP and IB4 in Grp ChR2 DRG. Scale bar in A , 50 μm. ( B ) eYFP image from Grp WT ; Ai32 DRG. Scale bar in B , 50 μm. ( C ) IHC image of eYFP, βlII-Tubulin and DAPI in Grp WT ; Ai32 nape skin. Scale bar in C , 100 μm. ( D ) IHC image of eYFP and ßIII-Tubulin in Grp ChR2 cheek skin. Scale bar in D , 100 μm. ( E ) IHC images of eYFP, CGRP and DAPI merge in Grp ChR2 glabrous skin. Scale bar in E , 100 μm. ( F ) IHC image of eYFP and DiI in Grp ChR2 DRG or TG 10 days after i.d. nape or cheek injection of DiI tracer. Scale bar in F , 50 μm.

Techniques Used: Immunohistochemistry, Injection

16) Product Images from "Reduced intraepithelial corneal nerve density and sensitivity accompany desiccating stress and aging in C57BL/6 mice"

Article Title: Reduced intraepithelial corneal nerve density and sensitivity accompany desiccating stress and aging in C57BL/6 mice

Journal: Experimental eye research

doi: 10.1016/j.exer.2018.01.024

Axon density and thickness are reduced in response to acute DS A. Representative images of a control and an acute DS cornea from d10 are presented. The ICNs have been visualized using an antibody against βIII tubulin. A minimum of 5 corneas for each variable were assessed. B. Images including those presented in 1A were used to quantify the axon density of the ICNs using Sholl analysis. The total number of corneas assessed (n) are indicated; both eyes of each animal were used. Each red or black circle represents the axon density of one cornea. Axon density decreased significantly relative to controls at d5 after MMC treatment and at d10 after both vehicle (veh) and MMC treatment. D . Representative images of corneas used to measure mean axon thickness are presented. E . Mean axon thickness was quantified and is presented. Axon thickness is reduced at d5 and d10 compared to controls. Bar in A = 500 µm; bar in D = 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Figure Legend Snippet: Axon density and thickness are reduced in response to acute DS A. Representative images of a control and an acute DS cornea from d10 are presented. The ICNs have been visualized using an antibody against βIII tubulin. A minimum of 5 corneas for each variable were assessed. B. Images including those presented in 1A were used to quantify the axon density of the ICNs using Sholl analysis. The total number of corneas assessed (n) are indicated; both eyes of each animal were used. Each red or black circle represents the axon density of one cornea. Axon density decreased significantly relative to controls at d5 after MMC treatment and at d10 after both vehicle (veh) and MMC treatment. D . Representative images of corneas used to measure mean axon thickness are presented. E . Mean axon thickness was quantified and is presented. Axon thickness is reduced at d5 and d10 compared to controls. Bar in A = 500 µm; bar in D = 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Techniques Used:

17) Product Images from "A tricyclic antidepressant, amoxapine, reduces amyloid-β generation through multiple serotonin receptor 6-mediated targets"

Article Title: A tricyclic antidepressant, amoxapine, reduces amyloid-β generation through multiple serotonin receptor 6-mediated targets

Journal: Scientific Reports

doi: 10.1038/s41598-017-04144-3

Amoxapine modulates Aβ generation in human neuronal cells differentiated from NSCs. ( A ) Representative image of neuronal differentiated human neural stem cells (NSCs) stained with Tuj1, Sox2, MAP2, GFAP or DAPI. Scale bar, 50 μm. ( B ) The mRNA level of HTR6 in human NSCs and NSC differentiated neuronal cells. ( C ) The levels of Aβ produced by neuronal differentiated NSCs in response to vehicle (0.1% DMSO), 10 μM BSI IV, 10 μM L685,458, or the indicated compounds at 3 μM or 10 μM for 24 hours. ( D ) The levels of Aβ produced by neuronal differentiated NSCs after treatment with vehicle (0.1% DMSO), amoxapine or SB258585 at 10 μM in the cells with the infection of scrambled or HTR6 gene-specific shRNA. Data are presented as the mean ± s.e.m. *p
Figure Legend Snippet: Amoxapine modulates Aβ generation in human neuronal cells differentiated from NSCs. ( A ) Representative image of neuronal differentiated human neural stem cells (NSCs) stained with Tuj1, Sox2, MAP2, GFAP or DAPI. Scale bar, 50 μm. ( B ) The mRNA level of HTR6 in human NSCs and NSC differentiated neuronal cells. ( C ) The levels of Aβ produced by neuronal differentiated NSCs in response to vehicle (0.1% DMSO), 10 μM BSI IV, 10 μM L685,458, or the indicated compounds at 3 μM or 10 μM for 24 hours. ( D ) The levels of Aβ produced by neuronal differentiated NSCs after treatment with vehicle (0.1% DMSO), amoxapine or SB258585 at 10 μM in the cells with the infection of scrambled or HTR6 gene-specific shRNA. Data are presented as the mean ± s.e.m. *p

Techniques Used: Staining, Produced, Infection, shRNA

18) Product Images from "Regenerative Drug Discovery Using Ear Pinna Punch Wound Model in Mice"

Article Title: Regenerative Drug Discovery Using Ear Pinna Punch Wound Model in Mice

Journal: Pharmaceuticals

doi: 10.3390/ph15050610

Development of nerve fibres and blood vessels in ear pinnae regenerating following zebularine and retinoic acid treatment on day 42 post-injury. Macroscopic images of ear pinnae used in the examination collected from mice ( a ) receiving saline, ( b ) treated with zebularine alone, and ( c ) treated with zebularine and retinoic acid. The areas selected for microscopic examination are indicated with white squares. ( e , h ) Microphotographs (10× objective lens) with immunohistochemical staining for neuron-specific class III ß-tubulin (Tuj1, light blue pseudocolour) and vascular smooth muscle (αSMA, red pseudocolour) of ear pinna wounds for saline-treated controls, ( f , i ) mice treated with zebularine alone, ( g , j ) and mice treated with zebularine and retinoic acid. The upper panels represent the outer ( e – g ) and lower ( h – j ) panels the inner aspects of the dissected ear pinnae. ( d ) A whole immunostained ear from the control under a lower magnification (5× objective lens) is shown for comparison.
Figure Legend Snippet: Development of nerve fibres and blood vessels in ear pinnae regenerating following zebularine and retinoic acid treatment on day 42 post-injury. Macroscopic images of ear pinnae used in the examination collected from mice ( a ) receiving saline, ( b ) treated with zebularine alone, and ( c ) treated with zebularine and retinoic acid. The areas selected for microscopic examination are indicated with white squares. ( e , h ) Microphotographs (10× objective lens) with immunohistochemical staining for neuron-specific class III ß-tubulin (Tuj1, light blue pseudocolour) and vascular smooth muscle (αSMA, red pseudocolour) of ear pinna wounds for saline-treated controls, ( f , i ) mice treated with zebularine alone, ( g , j ) and mice treated with zebularine and retinoic acid. The upper panels represent the outer ( e – g ) and lower ( h – j ) panels the inner aspects of the dissected ear pinnae. ( d ) A whole immunostained ear from the control under a lower magnification (5× objective lens) is shown for comparison.

Techniques Used: Mouse Assay, Immunohistochemistry, Staining

19) Product Images from "Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy"

Article Title: Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy

Journal: Scientific Reports

doi: 10.1038/s41598-020-63418-5

Effect of Vectashield (VS) on the fluorescence of AF647- and AF488-labelled microtubules. ( a ) Widefield images of ND7/23 cells labelled with AF647-conjugated anti-tubulin β3 (AF647-TUBB3) antibody in PBS (upper panels) and in VS (lower panels). To ensure the quenched AF647 signal in VS is visible, the same images are shown on the right with different linearly adjusted brightness and contrast levels (as indicated by look-up table (LUT) intensity scale bars). ( b ) Widefield images of ND7/23 cells labelled with AF488-conjugated anti-tubulin β3 (AF488-TUBB3) antibody in PBS (upper panel) and in VS (lower panel). Brightness and contrast were linearly adjusted to show the same display range in both PBS and VS conditions (as indicated by LUT intensity scale bars).
Figure Legend Snippet: Effect of Vectashield (VS) on the fluorescence of AF647- and AF488-labelled microtubules. ( a ) Widefield images of ND7/23 cells labelled with AF647-conjugated anti-tubulin β3 (AF647-TUBB3) antibody in PBS (upper panels) and in VS (lower panels). To ensure the quenched AF647 signal in VS is visible, the same images are shown on the right with different linearly adjusted brightness and contrast levels (as indicated by look-up table (LUT) intensity scale bars). ( b ) Widefield images of ND7/23 cells labelled with AF488-conjugated anti-tubulin β3 (AF488-TUBB3) antibody in PBS (upper panel) and in VS (lower panel). Brightness and contrast were linearly adjusted to show the same display range in both PBS and VS conditions (as indicated by LUT intensity scale bars).

Techniques Used: Fluorescence

20) Product Images from "Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington’s disease"

Article Title: Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington’s disease

Journal: Nature Communications

doi: 10.1038/s41467-021-25651-y

CHIR99021 neuroprotection in HD patient neurons requires CAST. a Knock-down efficiency of CAST and calpain I ( CAPN1 ) short hairpin RNA (shRNA) was confirmed by western blotting. b Mitochondrial membrane potential (MMP) was assessed using a TMRM fluorescence probe in control shRNA (shCon) or CAST shRNA (shCAST) expressing cells treated with CHIR99021 (chir) or vehicle (veh) ( n = 4). c Cell viability was assessed by MTT assay in shCon- or shCAST-expressing cells treated with CHIR99021 or vehicle ( n = 6). d MMP was measured by TMRM staining in control shRNA (shCon)- or sh CAPN1 -expressing cells treated with CHIR99021 or vehicle ( n = 4). e Cell viability was measured by MTT assay in shCon or sh CAPN1 cells treated with CHIR99021 or vehicle ( n = 8). Mixed striatal neurons were differentiated from iPS cells of patients with HD and normal subjects. Twenty days after neuronal differentiation, neurons were infected with lentivirus expressing GFP-labeled shCon or shCAST. f Knock-down efficiency of shCAST in neurons derived from HD patient iPS cells was confirmed by western blotting. Two days after infection, neurons derived from iPS cells from normal subjects (Nor) and HD patients (HD) were treated with CHIR99021 at 1 μM for 5 consecutive days. g MMP was assessed using a TMRM fluorescence probe ( n = 4). Cell viability was measured by MTT assay after brain-derived neurotrophic factor (BDNF) withdrawal for 12 h ( n = 4). h Neurons were stained with anti-MAP2 (red)/anti-GAD67 (blue) or anti-β-tubulin (clone Tuj1) (blue)/anti-DARPP-32 (red) to indicate dendritic and axonal morphology, respectively. Scale bar = 30 μm. i Quantification of MAP2 + neuronal dendrite length ( n = 23 cells/group). j Quantification of Tuj1 + neuronal axon length ( n = 28 cell/group). All values are reported as mean ± SEM and compared using one-way ANOVA with Tukey’s post hoc test. Data are representative of at least three independent experiments. Exact p values are shown in the figures.
Figure Legend Snippet: CHIR99021 neuroprotection in HD patient neurons requires CAST. a Knock-down efficiency of CAST and calpain I ( CAPN1 ) short hairpin RNA (shRNA) was confirmed by western blotting. b Mitochondrial membrane potential (MMP) was assessed using a TMRM fluorescence probe in control shRNA (shCon) or CAST shRNA (shCAST) expressing cells treated with CHIR99021 (chir) or vehicle (veh) ( n = 4). c Cell viability was assessed by MTT assay in shCon- or shCAST-expressing cells treated with CHIR99021 or vehicle ( n = 6). d MMP was measured by TMRM staining in control shRNA (shCon)- or sh CAPN1 -expressing cells treated with CHIR99021 or vehicle ( n = 4). e Cell viability was measured by MTT assay in shCon or sh CAPN1 cells treated with CHIR99021 or vehicle ( n = 8). Mixed striatal neurons were differentiated from iPS cells of patients with HD and normal subjects. Twenty days after neuronal differentiation, neurons were infected with lentivirus expressing GFP-labeled shCon or shCAST. f Knock-down efficiency of shCAST in neurons derived from HD patient iPS cells was confirmed by western blotting. Two days after infection, neurons derived from iPS cells from normal subjects (Nor) and HD patients (HD) were treated with CHIR99021 at 1 μM for 5 consecutive days. g MMP was assessed using a TMRM fluorescence probe ( n = 4). Cell viability was measured by MTT assay after brain-derived neurotrophic factor (BDNF) withdrawal for 12 h ( n = 4). h Neurons were stained with anti-MAP2 (red)/anti-GAD67 (blue) or anti-β-tubulin (clone Tuj1) (blue)/anti-DARPP-32 (red) to indicate dendritic and axonal morphology, respectively. Scale bar = 30 μm. i Quantification of MAP2 + neuronal dendrite length ( n = 23 cells/group). j Quantification of Tuj1 + neuronal axon length ( n = 28 cell/group). All values are reported as mean ± SEM and compared using one-way ANOVA with Tukey’s post hoc test. Data are representative of at least three independent experiments. Exact p values are shown in the figures.

Techniques Used: shRNA, Western Blot, Fluorescence, Expressing, MTT Assay, Staining, Infection, Labeling, Derivative Assay

21) Product Images from "SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis"

Article Title: SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis

Journal: Molecular Neurodegeneration

doi: 10.1186/s13024-021-00475-y

Generation of whole-cell and cytoplasmic transcriptomes from healthy and C9ORF72-ALS patient-derived neurons. A Three healthy control and three C9ORF72-ALS (C9-ALS) lines of patient-derived neurons were treated with Ctrl-RNAi (C-RNAi) or SRSF1-RNAi (ΔSRSF1) prior to whole-cell (T) lysis or nuclear (N) and cytoplasmic (C) fractionation. Western blots were probed for the nuclear chromatin remodelling SSRP1 factor and the neuronal cytoplasmic marker TUJ1. B Relative expression levels of SRSF1 mRNA in whole-cell patient-derived neurons prepared in A were quantified using qRT-PCR in biological triplicates following normalization to U1 snRNA levels and to 100% for healthy neurons treated with C-RNAi (mean ± SEM; one-way ANOVA with Tukey’s correction for multiple comparisons, **: p
Figure Legend Snippet: Generation of whole-cell and cytoplasmic transcriptomes from healthy and C9ORF72-ALS patient-derived neurons. A Three healthy control and three C9ORF72-ALS (C9-ALS) lines of patient-derived neurons were treated with Ctrl-RNAi (C-RNAi) or SRSF1-RNAi (ΔSRSF1) prior to whole-cell (T) lysis or nuclear (N) and cytoplasmic (C) fractionation. Western blots were probed for the nuclear chromatin remodelling SSRP1 factor and the neuronal cytoplasmic marker TUJ1. B Relative expression levels of SRSF1 mRNA in whole-cell patient-derived neurons prepared in A were quantified using qRT-PCR in biological triplicates following normalization to U1 snRNA levels and to 100% for healthy neurons treated with C-RNAi (mean ± SEM; one-way ANOVA with Tukey’s correction for multiple comparisons, **: p

Techniques Used: Derivative Assay, Lysis, Fractionation, Western Blot, Marker, Expressing, Quantitative RT-PCR

22) Product Images from "Disrupted Association of Sensory Neurons With Enveloping Satellite Glial Cells in Fragile X Mouse Model"

Article Title: Disrupted Association of Sensory Neurons With Enveloping Satellite Glial Cells in Fragile X Mouse Model

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2021.796070

Molecular changes in Fmr1 KO small/medium sensory neurons indicate delayed differentiation. (A) Volcano plot of DE genes in small/medium neurons from Fmr1 KO compared to WT. (B) Enriched pathways in Fmr1 KO compared to WT (GO Biological Process) for up-regulated (red) and down-regulated (blue) genes. (C) Mean expression of the neuronal markers Nefl , Prph , Tubb3 , and Calca in WT (black) and Fmr1 KO (red). (D) qPCR analysis of the relative mRNA expression of Tubb3 , Nefl , and Stmn2 in Fmr1 KO compared to WT DRGs. n = 3 biologically independent animals.
Figure Legend Snippet: Molecular changes in Fmr1 KO small/medium sensory neurons indicate delayed differentiation. (A) Volcano plot of DE genes in small/medium neurons from Fmr1 KO compared to WT. (B) Enriched pathways in Fmr1 KO compared to WT (GO Biological Process) for up-regulated (red) and down-regulated (blue) genes. (C) Mean expression of the neuronal markers Nefl , Prph , Tubb3 , and Calca in WT (black) and Fmr1 KO (red). (D) qPCR analysis of the relative mRNA expression of Tubb3 , Nefl , and Stmn2 in Fmr1 KO compared to WT DRGs. n = 3 biologically independent animals.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

23) Product Images from "GSK3B-mediated phosphorylation of MCL1 regulates axonal autophagy to promote Wallerian degeneration"

Article Title: GSK3B-mediated phosphorylation of MCL1 regulates axonal autophagy to promote Wallerian degeneration

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201606020

Inhibition of the GSK3B–MCL1 pathway delays the Wallerian degeneration of optic nerves in vivo. A unilateral intravitreal injection of adenoviral vector solution for the expression of the indicated molecules in retinal ganglion neurons was performed in adult mice, followed by optic nerve transection 5 d after the injection to induce Wallerian degeneration. Degeneration of the optic nerve (between the eye and optic chiasm) was assessed by the expression of neurofilament M at the indicated time points. DAS, days after surgery. (A) Representative photomicrographs for the immunostaining of neurofilament M on longitudinal optic nerve sections are shown. Bar, 25 µm. (B) Quantified immunofluorescent intensity for each condition relative to the intensity of a “no-axotomy” sample is shown (mean ± SEM, n = 5). (C) Representative immunoblots for the expression of the indicated molecules in the optic nerves 5 d after infection is shown. (D) Representative immunoblot for the expression of neurofilament M for each condition is shown. β-Actin served as a loading control. (E) Quantified levels of neurofilament M normalized to β-actin at the indicated time points are shown relative to the no-axotomy condition (mean ± SEM, n = 5). We obtained essentially the same results by using βIII-tubulin as a marker for intact axons (not depicted). Significant differences from the control (**, P
Figure Legend Snippet: Inhibition of the GSK3B–MCL1 pathway delays the Wallerian degeneration of optic nerves in vivo. A unilateral intravitreal injection of adenoviral vector solution for the expression of the indicated molecules in retinal ganglion neurons was performed in adult mice, followed by optic nerve transection 5 d after the injection to induce Wallerian degeneration. Degeneration of the optic nerve (between the eye and optic chiasm) was assessed by the expression of neurofilament M at the indicated time points. DAS, days after surgery. (A) Representative photomicrographs for the immunostaining of neurofilament M on longitudinal optic nerve sections are shown. Bar, 25 µm. (B) Quantified immunofluorescent intensity for each condition relative to the intensity of a “no-axotomy” sample is shown (mean ± SEM, n = 5). (C) Representative immunoblots for the expression of the indicated molecules in the optic nerves 5 d after infection is shown. (D) Representative immunoblot for the expression of neurofilament M for each condition is shown. β-Actin served as a loading control. (E) Quantified levels of neurofilament M normalized to β-actin at the indicated time points are shown relative to the no-axotomy condition (mean ± SEM, n = 5). We obtained essentially the same results by using βIII-tubulin as a marker for intact axons (not depicted). Significant differences from the control (**, P

Techniques Used: Inhibition, In Vivo, Injection, Plasmid Preparation, Expressing, Mouse Assay, Immunostaining, Western Blot, Infection, Marker

Down-regulated expression of fbxw7 prevents autophagy and axonal degeneration. (A and B) The down-regulated expression of atg5 and atg7 by the two independent shRNAs used in this study was confirmed by quantitative RT-PCR (A) and immunoblot analysis (B). The expression level of each molecule normalized to β-actin is shown relative to that of the no-infection condition in A (mean ± SEM, n = 5). Representative immunoblots for the analysis of lysates prepared from axons expressing shRNA against atg5 and atg7 are shown in B. β-Actin serves as a loading control. The expression level of each molecule normalized to β-actin relative to that of the nontarget control shRNA-expressing condition is also shown (mean ± SEM, n = 3). (C and D) Axonal autophagy activity with the shRNA-mediated down-regulation of fbxw7 expression was assessed using an in vitro Wallerian degeneration model. Representative photomicrographs for GFP and immunostaining of β-tubulin in degenerating axons of cultured GFP-LC3B Tg DRG neurons are shown in C. Bar, 10 µm. Lysates prepared from axons were subjected to immunoblot analysis using the indicated antibodies. Representative immunoblots (top) and quantified expression levels for GFP-LC3B-II (middle) normalized to GFP-LC3B-I or p62 (bottom) normalized to β-actin relative to the control are shown in D (mean ± SEM, n = 5). (E–H) Down-regulation of expression of key Atg genes prevents axonal autophagy. Axonal degeneration with shRNA-mediated down-regulation of atg5 , atg7 , or fbxw7 expression was assessed by immunocytochemistry for β-tubulin (E and F) and immunoblot for neurofilament M (G and H) using an in vitro Wallerian degeneration model. Nontarget control shRNA-infected neurons served as a negative control. Representative photomicrographs of axons are shown in E. Bar, 20 µm. Axonal degeneration index value calculated for each condition at the indicated time point after transection is shown in F. (G and H) Lysates were prepared from axons at the indicated time points after transection and analyzed by immunoblot. Representative immunoblots using the indicated antibodies and relative expression levels for neurofilament M normalized to β-actin are shown (mean ± SEM, n = 5). Significant differences from the control (*, P
Figure Legend Snippet: Down-regulated expression of fbxw7 prevents autophagy and axonal degeneration. (A and B) The down-regulated expression of atg5 and atg7 by the two independent shRNAs used in this study was confirmed by quantitative RT-PCR (A) and immunoblot analysis (B). The expression level of each molecule normalized to β-actin is shown relative to that of the no-infection condition in A (mean ± SEM, n = 5). Representative immunoblots for the analysis of lysates prepared from axons expressing shRNA against atg5 and atg7 are shown in B. β-Actin serves as a loading control. The expression level of each molecule normalized to β-actin relative to that of the nontarget control shRNA-expressing condition is also shown (mean ± SEM, n = 3). (C and D) Axonal autophagy activity with the shRNA-mediated down-regulation of fbxw7 expression was assessed using an in vitro Wallerian degeneration model. Representative photomicrographs for GFP and immunostaining of β-tubulin in degenerating axons of cultured GFP-LC3B Tg DRG neurons are shown in C. Bar, 10 µm. Lysates prepared from axons were subjected to immunoblot analysis using the indicated antibodies. Representative immunoblots (top) and quantified expression levels for GFP-LC3B-II (middle) normalized to GFP-LC3B-I or p62 (bottom) normalized to β-actin relative to the control are shown in D (mean ± SEM, n = 5). (E–H) Down-regulation of expression of key Atg genes prevents axonal autophagy. Axonal degeneration with shRNA-mediated down-regulation of atg5 , atg7 , or fbxw7 expression was assessed by immunocytochemistry for β-tubulin (E and F) and immunoblot for neurofilament M (G and H) using an in vitro Wallerian degeneration model. Nontarget control shRNA-infected neurons served as a negative control. Representative photomicrographs of axons are shown in E. Bar, 20 µm. Axonal degeneration index value calculated for each condition at the indicated time point after transection is shown in F. (G and H) Lysates were prepared from axons at the indicated time points after transection and analyzed by immunoblot. Representative immunoblots using the indicated antibodies and relative expression levels for neurofilament M normalized to β-actin are shown (mean ± SEM, n = 5). Significant differences from the control (*, P

Techniques Used: Expressing, Quantitative RT-PCR, Infection, Western Blot, shRNA, Activity Assay, In Vitro, Immunostaining, Cell Culture, Immunocytochemistry, Negative Control

GSK3B-mediated phosphorylation of MCL1 leads to activation of axonal autophagy. (A and B) The GSK3B–MCL1 pathway regulates the induction of autophagy in transected axons. Axonal autophagic activity was assessed in degenerating axons in vitro using cultured DRG neurons from GFP-LC3B Tg mice. Representative photomicrographs for GFP and immunostaining against β-tubulin (red) of degenerating axons expressing the indicated molecules are shown in A. Bar, 10 µm. Lysates were prepared from cultured GFP-LC3B Tg DRG axons before and 3 h after transection. Representative immunoblots and quantified levels for GFP-LC3II normalized to GFP-LC3B-I relative to the control (open bar) are shown (mean ± SEM, n = 5) in B. (C and D) Inhibition of the GSK3B–MCL1 pathway prevents autophagosome-like vacuole formation in the degenerating optic nerve axons in vivo. A unilateral intravitreal injection of adenoviral vector solution for the expression of the indicated molecules in retinal ganglion neurons was performed in adult mice, followed by optic nerve transection 5 d after the injection to induce Wallerian degeneration. Autophagosome formation in optic nerve axons (between the eye and optic chiasm) was assessed by electron microscopy at the indicated time points. Representative photomicrographs for an electron microscopic analysis of transverse optic nerve sections are shown in C. Arrowheads indicate autophagosome-like vacuoles in C. Bar, 1 µm. The number of autophagosomes to optic nerve axons under each condition is shown in (D; mean ± SEM, n = 3). Significant differences from the control (*, P
Figure Legend Snippet: GSK3B-mediated phosphorylation of MCL1 leads to activation of axonal autophagy. (A and B) The GSK3B–MCL1 pathway regulates the induction of autophagy in transected axons. Axonal autophagic activity was assessed in degenerating axons in vitro using cultured DRG neurons from GFP-LC3B Tg mice. Representative photomicrographs for GFP and immunostaining against β-tubulin (red) of degenerating axons expressing the indicated molecules are shown in A. Bar, 10 µm. Lysates were prepared from cultured GFP-LC3B Tg DRG axons before and 3 h after transection. Representative immunoblots and quantified levels for GFP-LC3II normalized to GFP-LC3B-I relative to the control (open bar) are shown (mean ± SEM, n = 5) in B. (C and D) Inhibition of the GSK3B–MCL1 pathway prevents autophagosome-like vacuole formation in the degenerating optic nerve axons in vivo. A unilateral intravitreal injection of adenoviral vector solution for the expression of the indicated molecules in retinal ganglion neurons was performed in adult mice, followed by optic nerve transection 5 d after the injection to induce Wallerian degeneration. Autophagosome formation in optic nerve axons (between the eye and optic chiasm) was assessed by electron microscopy at the indicated time points. Representative photomicrographs for an electron microscopic analysis of transverse optic nerve sections are shown in C. Arrowheads indicate autophagosome-like vacuoles in C. Bar, 1 µm. The number of autophagosomes to optic nerve axons under each condition is shown in (D; mean ± SEM, n = 3). Significant differences from the control (*, P

Techniques Used: Activation Assay, Activity Assay, In Vitro, Cell Culture, Mouse Assay, Immunostaining, Expressing, Western Blot, Inhibition, In Vivo, Injection, Plasmid Preparation, Electron Microscopy

FBXW7 targets MCL1 pS140 on mitochondria. (A and B) The down-regulated expression of fbxw7 inhibits Wallerian degeneration. The degeneration of axons expressing the indicated shRNA was assessed by an in vitro Wallerian degeneration model. Representative photomicrographs for β-tubulin immunostaining of axons are shown in A. Bar, 50 µm. The axonal degeneration index value calculated for each condition 24 h after the induction of degeneration is shown in B. (C and D) The down-regulated expression of fbxw7 results in an increase in the expression and decrease in the polyubiquitinated (Ub) levels of MCL1 in transected axons. Lysates were prepared from the axons expressing shRNA against fbxw7 . Representative immunoblots (C) and immunoprecipitation (D) using an anti-MCL1 antibody analyzed by immunoblotting and quantified levels for MCL1 or polyubiquitinated MCL1 normalized to β-actin relative to the control are shown. (E) The down-regulated expression of fbxw7 stabilizes MCL1. Representative immunoblots and quantified expression levels of MCL1 normalized to β-actin relative to the control (open bar) are shown (mean ± SEM, n = 3). (F) The down-regulated expression of fbxw7 inhibits MCL1 release from mitochondria. Mitochondrial fractions were prepared from axons expressing shRNA against fbxw7 . Representative immunoblots (top) and quantified expression levels of MCL1 normalized to prohibitin (middle) or MCL1 pS140 normalized to MCL1 (bottom) relative to the control (open bar) in mitochondrial fractions are shown (mean ± SEM, n = 5). (G) Demonstration of the direct interaction between FBXW7 and MCL1 in murine neuroblastoma Neuro-2a cells. Representative immunoblots (IB) and immunoprecipitation using an anti-FLAG antibody analyzed by immunoblotting and quantified levels for MCL1-HA normalized to FLAG-FBXW7 relative to the control (open bar) are shown (mean ± SEM, n = 5). Significant differences from the control (*, P
Figure Legend Snippet: FBXW7 targets MCL1 pS140 on mitochondria. (A and B) The down-regulated expression of fbxw7 inhibits Wallerian degeneration. The degeneration of axons expressing the indicated shRNA was assessed by an in vitro Wallerian degeneration model. Representative photomicrographs for β-tubulin immunostaining of axons are shown in A. Bar, 50 µm. The axonal degeneration index value calculated for each condition 24 h after the induction of degeneration is shown in B. (C and D) The down-regulated expression of fbxw7 results in an increase in the expression and decrease in the polyubiquitinated (Ub) levels of MCL1 in transected axons. Lysates were prepared from the axons expressing shRNA against fbxw7 . Representative immunoblots (C) and immunoprecipitation (D) using an anti-MCL1 antibody analyzed by immunoblotting and quantified levels for MCL1 or polyubiquitinated MCL1 normalized to β-actin relative to the control are shown. (E) The down-regulated expression of fbxw7 stabilizes MCL1. Representative immunoblots and quantified expression levels of MCL1 normalized to β-actin relative to the control (open bar) are shown (mean ± SEM, n = 3). (F) The down-regulated expression of fbxw7 inhibits MCL1 release from mitochondria. Mitochondrial fractions were prepared from axons expressing shRNA against fbxw7 . Representative immunoblots (top) and quantified expression levels of MCL1 normalized to prohibitin (middle) or MCL1 pS140 normalized to MCL1 (bottom) relative to the control (open bar) in mitochondrial fractions are shown (mean ± SEM, n = 5). (G) Demonstration of the direct interaction between FBXW7 and MCL1 in murine neuroblastoma Neuro-2a cells. Representative immunoblots (IB) and immunoprecipitation using an anti-FLAG antibody analyzed by immunoblotting and quantified levels for MCL1-HA normalized to FLAG-FBXW7 relative to the control (open bar) are shown (mean ± SEM, n = 5). Significant differences from the control (*, P

Techniques Used: Expressing, shRNA, In Vitro, Immunostaining, Western Blot, Immunoprecipitation

MCL1 phosphorylation at S140 is involved in the progression of Wallerian degeneration. (A and B) Axonal protective effects induced by the expression of the indicated genes were assessed by in vitro Wallerian degeneration experiments using axons from cultured DRG explant neurons. Representative photomicrographs of immunostaining against β-tubulin of degenerating axons 24 h after transection are shown in A. Bar, 25 µm. The axonal degeneration index value calculated for each condition at the indicated time point is shown in B (mean ± SEM, n = 5). (C) MCL1 is phosphorylated at S140 during Wallerian degeneration. Lysates were prepared from axons at the indicated time points after transection. Representative immunoblots and quantified expression levels for MCL1 pS140 normalized to the MCL1 level relative to the control (open bar; mean ± SEM, n = 5) are shown. Significant differences from the control (*, P
Figure Legend Snippet: MCL1 phosphorylation at S140 is involved in the progression of Wallerian degeneration. (A and B) Axonal protective effects induced by the expression of the indicated genes were assessed by in vitro Wallerian degeneration experiments using axons from cultured DRG explant neurons. Representative photomicrographs of immunostaining against β-tubulin of degenerating axons 24 h after transection are shown in A. Bar, 25 µm. The axonal degeneration index value calculated for each condition at the indicated time point is shown in B (mean ± SEM, n = 5). (C) MCL1 is phosphorylated at S140 during Wallerian degeneration. Lysates were prepared from axons at the indicated time points after transection. Representative immunoblots and quantified expression levels for MCL1 pS140 normalized to the MCL1 level relative to the control (open bar; mean ± SEM, n = 5) are shown. Significant differences from the control (*, P

Techniques Used: Expressing, In Vitro, Cell Culture, Immunostaining, Western Blot

Inhibition of autophagy in optic nerve axons deteriorates the recruitment of phagocytes to axonal debris . A unilateral intravitreal injection of adenoviral vector solution for the expression of the indicated molecules in retinal ganglion neurons was performed in adult mice, followed by optic nerve transection 5 d after the injection to induce Wallerian degeneration. The degeneration of the optic nerve (between the eye and optic chiasm) was assessed by immunostaining using antibodies against βIII-tubulin (red) and F4/80 (green) 2 d after axotomy. Nuclei were counterstained with DAPI. Representative photomicrographs for the immunostaining of longitudinal optic nerve sections are shown in A. Bar, 25 µm. Immunofluorescent intensity ratio of the F4/80-stained axonal area to the total axonal area in each condition relative to that of the nontarget control shRNA-expressing condition, representing the relative number of phagocytic cells in axons under each condition, is shown in B (mean ± SEM, n = 3). The nontarget control shRNA-expressing condition and Cre-only expression serve as negative controls for the shRNA-mediated down-regulation and overexpression of the indicated molecules, respectively. Significant differences from the control (**, P
Figure Legend Snippet: Inhibition of autophagy in optic nerve axons deteriorates the recruitment of phagocytes to axonal debris . A unilateral intravitreal injection of adenoviral vector solution for the expression of the indicated molecules in retinal ganglion neurons was performed in adult mice, followed by optic nerve transection 5 d after the injection to induce Wallerian degeneration. The degeneration of the optic nerve (between the eye and optic chiasm) was assessed by immunostaining using antibodies against βIII-tubulin (red) and F4/80 (green) 2 d after axotomy. Nuclei were counterstained with DAPI. Representative photomicrographs for the immunostaining of longitudinal optic nerve sections are shown in A. Bar, 25 µm. Immunofluorescent intensity ratio of the F4/80-stained axonal area to the total axonal area in each condition relative to that of the nontarget control shRNA-expressing condition, representing the relative number of phagocytic cells in axons under each condition, is shown in B (mean ± SEM, n = 3). The nontarget control shRNA-expressing condition and Cre-only expression serve as negative controls for the shRNA-mediated down-regulation and overexpression of the indicated molecules, respectively. Significant differences from the control (**, P

Techniques Used: Inhibition, Injection, Plasmid Preparation, Expressing, Mouse Assay, Immunostaining, Staining, shRNA, Over Expression

The GSK3B–MCL1 pathway is involved in injury-induced axonal autophagy. (A–C) The down-regulated expression of mcl1 by specific shRNA in cultured DRG neurons by the two independent shRNAs used in this study was confirmed by quantitative RT-PCR (A) and immunoblot analysis (B and C). Expression levels of mcl1 normalized to β-actin relative to those of the nontarget control shRNA-expressing condition are shown in A (mean ± SEM, n = 5). Lysates prepared from the axons expressing the indicated molecules were examined by immunoblot analysis using the indicated antibodies. Representative images of immunoblots (C) and quantified expression level of MCL1 normalized to β-actin relative to that of the nontarget control shRNA-expressing condition (B) are shown. (D and E) Axonal autophagic activity during Wallerian degeneration in vitro was assessed using cultured GFP-LC3B Tg DRG neurons. Representative photomicrographs for GFP and immunostaining against β-tubulin (red) of degenerating axons expressing the indicated molecules are shown in D. Bar, 10 µm. Lysates prepared from the axons of cultured GFP-LC3B Tg DRG neurons were subjected to immunoblot analysis using the indicated antibodies. Representative immunoblots and quantified levels for GFP-LC3B-II normalized to GFP-LC3B-I relative to the control (open bar) are shown in E (mean ± SEM, n = 5). Note that the reduced expression of MCL1 in neurons expressing shRNA for MCL1 was rescued by the expression of shRNA-resistant MCL1. Significant differences from the control (**, P
Figure Legend Snippet: The GSK3B–MCL1 pathway is involved in injury-induced axonal autophagy. (A–C) The down-regulated expression of mcl1 by specific shRNA in cultured DRG neurons by the two independent shRNAs used in this study was confirmed by quantitative RT-PCR (A) and immunoblot analysis (B and C). Expression levels of mcl1 normalized to β-actin relative to those of the nontarget control shRNA-expressing condition are shown in A (mean ± SEM, n = 5). Lysates prepared from the axons expressing the indicated molecules were examined by immunoblot analysis using the indicated antibodies. Representative images of immunoblots (C) and quantified expression level of MCL1 normalized to β-actin relative to that of the nontarget control shRNA-expressing condition (B) are shown. (D and E) Axonal autophagic activity during Wallerian degeneration in vitro was assessed using cultured GFP-LC3B Tg DRG neurons. Representative photomicrographs for GFP and immunostaining against β-tubulin (red) of degenerating axons expressing the indicated molecules are shown in D. Bar, 10 µm. Lysates prepared from the axons of cultured GFP-LC3B Tg DRG neurons were subjected to immunoblot analysis using the indicated antibodies. Representative immunoblots and quantified levels for GFP-LC3B-II normalized to GFP-LC3B-I relative to the control (open bar) are shown in E (mean ± SEM, n = 5). Note that the reduced expression of MCL1 in neurons expressing shRNA for MCL1 was rescued by the expression of shRNA-resistant MCL1. Significant differences from the control (**, P

Techniques Used: Expressing, shRNA, Cell Culture, Quantitative RT-PCR, Western Blot, Activity Assay, In Vitro, Immunostaining

24) Product Images from "Diurnal Control of Sensory Axon Growth and Shedding in the Mouse Cornea"

Article Title: Diurnal Control of Sensory Axon Growth and Shedding in the Mouse Cornea

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.61.11.1

Axon density, assessed by Sholl analysis, also varies as function of time. ( A) Representative image taken of a female cornea from a mouse sacrificed at ZT 10.5. The cornea was stained with antibodies against βIII tubulin to reveal the ICNs. Each cornea was imaged using a spinning disk microscope with 7 × 3 individual images stitched together as described in the methods section. Scale bar: 500 µm. ( B) Axon density was assessed in 10 male and 10 female mice at ZT 22.5, ZT 1.5, ZT 10.5, and ZT 13.5 using Sholl analysis. The data shown were obtained by assessing axon density at four sites at the corneal periphery and three sites in the corneal center for males and females and after combining data from both sexes. Axon density is maximal at ZT 22.5 and lower 90 min before and 90 min after lights are turned off. In both sexes, axon density is greatest at ZT 22.5 and lowest between ZT 1.5 and ZT 13.5.
Figure Legend Snippet: Axon density, assessed by Sholl analysis, also varies as function of time. ( A) Representative image taken of a female cornea from a mouse sacrificed at ZT 10.5. The cornea was stained with antibodies against βIII tubulin to reveal the ICNs. Each cornea was imaged using a spinning disk microscope with 7 × 3 individual images stitched together as described in the methods section. Scale bar: 500 µm. ( B) Axon density was assessed in 10 male and 10 female mice at ZT 22.5, ZT 1.5, ZT 10.5, and ZT 13.5 using Sholl analysis. The data shown were obtained by assessing axon density at four sites at the corneal periphery and three sites in the corneal center for males and females and after combining data from both sexes. Axon density is maximal at ZT 22.5 and lower 90 min before and 90 min after lights are turned off. In both sexes, axon density is greatest at ZT 22.5 and lowest between ZT 1.5 and ZT 13.5.

Techniques Used: Staining, Microscopy, Mouse Assay

Localization of axonal proteins βIII tubulin and L1CAM in the mouse ICBNs and ICNTs visualized using 3D confocal imaging as a function of the time mice are euthanized. Four corneas from 2 male and 2 female mice euthanized at ZT 22.5, ZT 1.5, ZT 10.5, and ZT 13.5 were processed to visualize βIII tubulin and L1CAM. At the far right, the regions indicated by the asterisks in the apical most images are enlarged 3x with the two colors presented individually. These data are quantified in Figure 3 . Scale bar: 25 µm
Figure Legend Snippet: Localization of axonal proteins βIII tubulin and L1CAM in the mouse ICBNs and ICNTs visualized using 3D confocal imaging as a function of the time mice are euthanized. Four corneas from 2 male and 2 female mice euthanized at ZT 22.5, ZT 1.5, ZT 10.5, and ZT 13.5 were processed to visualize βIII tubulin and L1CAM. At the far right, the regions indicated by the asterisks in the apical most images are enlarged 3x with the two colors presented individually. These data are quantified in Figure 3 . Scale bar: 25 µm

Techniques Used: Imaging, Mouse Assay

Localization and expression of tight junction protein ZO1 and terminal differentiation protein IVL are altered 90 minutes before lights are turned on. (A) Shown are representative en face confocal images projecting through the apical most 10 µm of the cornea that was stained to show βIII tubulin ( red ), ZO1 ( green ), involucrin ( magenta ), and DAPI ( blue ). ZO1 is expressed at cell/cell borders at ZT 22.5 but areas are seen where it is disrupted. While at all other time points ZO1 is expressed uniformly in all apical cells, by ZT 13.5, expression is reduced. While the cytoplasm of the majority of the apical cells contain involucrin, there are more cells that do not express detectible IVL at ZT 22.5 compared to all other time points. Scale bar for en face image A: 25 µm; ( B) The 3D cross-sections projecting through 135 µm of the tissue were generated showing the apical 10 µm of the cornea. The involucrin+ cells are also surrounded by ZO1+ cell membranes and at ZT 22.5, several ICNTs insert between the apical squames ( arrows ). By contrast, at ZT 1.5 ICNTs do not penetrate the apical cell layer . At ZT 13.5, a single ICNT is seen that penetrates all the way to the apical cell layer. Scale bar for cross-sectional image B : 10 µm.
Figure Legend Snippet: Localization and expression of tight junction protein ZO1 and terminal differentiation protein IVL are altered 90 minutes before lights are turned on. (A) Shown are representative en face confocal images projecting through the apical most 10 µm of the cornea that was stained to show βIII tubulin ( red ), ZO1 ( green ), involucrin ( magenta ), and DAPI ( blue ). ZO1 is expressed at cell/cell borders at ZT 22.5 but areas are seen where it is disrupted. While at all other time points ZO1 is expressed uniformly in all apical cells, by ZT 13.5, expression is reduced. While the cytoplasm of the majority of the apical cells contain involucrin, there are more cells that do not express detectible IVL at ZT 22.5 compared to all other time points. Scale bar for en face image A: 25 µm; ( B) The 3D cross-sections projecting through 135 µm of the tissue were generated showing the apical 10 µm of the cornea. The involucrin+ cells are also surrounded by ZO1+ cell membranes and at ZT 22.5, several ICNTs insert between the apical squames ( arrows ). By contrast, at ZT 1.5 ICNTs do not penetrate the apical cell layer . At ZT 13.5, a single ICNT is seen that penetrates all the way to the apical cell layer. Scale bar for cross-sectional image B : 10 µm.

Techniques Used: Expressing, Staining, Generated

Changes in localization of βIII tubulin, GAP43, and L1CAM take place over time. (A) The expression of βIII tubulin in ICBNs is maximal 90 minutes before the time when lights are turned on at ZT 22.5; thereafter, βIII tubulin expression remains the same in the ICBNs. GAP43 expression in the ICBNs does not change significantly over time. The ratio of GAP43/βIII tubulin is lowest at ZT 22.5 and higher at all other time points indicating that axons are actively growing while the lights are on and continue to grow for 90 minutes after the lights are turned off at ZT 13.5. (B) ICNTs in the middle layers of the corneal epithelium appear as puncta. Significant differences are seen between ZT 22.5 and ZT 1.5 and between ZT 1.5 and ZT 13.5 for βIII tubulin. No significant differences are seen in GAP43; the ratio of GAP43/βIII tubulin in the ICNTs is highest at ZT 1.5. (C) In the apical aspect of the cornea where the ICNTs turn and assume a parallel orientation, there is a significant drop in the lengths of the pICNTs between ZT 22.5 and ZT 1.5 that is observed when the lengths of the pICNTs are quantified using βIII tubulin and GAP43; this is not observed when the lengths of the pICNTs are assessed using L1CAM.
Figure Legend Snippet: Changes in localization of βIII tubulin, GAP43, and L1CAM take place over time. (A) The expression of βIII tubulin in ICBNs is maximal 90 minutes before the time when lights are turned on at ZT 22.5; thereafter, βIII tubulin expression remains the same in the ICBNs. GAP43 expression in the ICBNs does not change significantly over time. The ratio of GAP43/βIII tubulin is lowest at ZT 22.5 and higher at all other time points indicating that axons are actively growing while the lights are on and continue to grow for 90 minutes after the lights are turned off at ZT 13.5. (B) ICNTs in the middle layers of the corneal epithelium appear as puncta. Significant differences are seen between ZT 22.5 and ZT 1.5 and between ZT 1.5 and ZT 13.5 for βIII tubulin. No significant differences are seen in GAP43; the ratio of GAP43/βIII tubulin in the ICNTs is highest at ZT 1.5. (C) In the apical aspect of the cornea where the ICNTs turn and assume a parallel orientation, there is a significant drop in the lengths of the pICNTs between ZT 22.5 and ZT 1.5 that is observed when the lengths of the pICNTs are quantified using βIII tubulin and GAP43; this is not observed when the lengths of the pICNTs are assessed using L1CAM.

Techniques Used: Expressing

Localization of axonal proteins βIII tubulin, GAP43, and LAMP1 in the mouse visualized in cross-sections using 3D confocal imaging as a function of time mice are euthanized. (A) En face confocal image stacks through the corneal epithelium were obtained and rotated to generate a cross sectional view. The images project through 135 µm of cornea epithelium to reveal the ICNTs projecting apically and occasionally turning to generate pICNTs. While LAMP1 is expressed most abundantly in the apical most cell layers and appears most abundant at 5:30 am, the wide dynamic range of its expression complicated obtaining representative images. Scale bar: 20 µm. (B) We quantified βIII tubulin and GAP43 pixel intensity in the middle of the epithelium in the ICNTs in cross-sectional images. The expression of βIII tubulin in ICNTs decreases significantly between ZT 22.5 and ZT 1.5 and remains the same at ZT 1.5, ZT 10.5, and ZT 13.5. The ratio of GAP43/ III tubulin increased significantly between ZT 22.5 and ZT 10.5. ( C) LAMP1 was quantified in five male and five female corneas. The expression of LAMP1 observed at ZT 22.5 was five- to sixfold higher than that seen at all other time points. By ZT 13.5, LAMP1 is at its lowest. By ZT 22.5, 90 minutes before the lights are turned on at ZT 0, LAMP1 expression is at its maximum.
Figure Legend Snippet: Localization of axonal proteins βIII tubulin, GAP43, and LAMP1 in the mouse visualized in cross-sections using 3D confocal imaging as a function of time mice are euthanized. (A) En face confocal image stacks through the corneal epithelium were obtained and rotated to generate a cross sectional view. The images project through 135 µm of cornea epithelium to reveal the ICNTs projecting apically and occasionally turning to generate pICNTs. While LAMP1 is expressed most abundantly in the apical most cell layers and appears most abundant at 5:30 am, the wide dynamic range of its expression complicated obtaining representative images. Scale bar: 20 µm. (B) We quantified βIII tubulin and GAP43 pixel intensity in the middle of the epithelium in the ICNTs in cross-sectional images. The expression of βIII tubulin in ICNTs decreases significantly between ZT 22.5 and ZT 1.5 and remains the same at ZT 1.5, ZT 10.5, and ZT 13.5. The ratio of GAP43/ III tubulin increased significantly between ZT 22.5 and ZT 10.5. ( C) LAMP1 was quantified in five male and five female corneas. The expression of LAMP1 observed at ZT 22.5 was five- to sixfold higher than that seen at all other time points. By ZT 13.5, LAMP1 is at its lowest. By ZT 22.5, 90 minutes before the lights are turned on at ZT 0, LAMP1 expression is at its maximum.

Techniques Used: Imaging, Mouse Assay, Expressing

Localization of axonal proteins βIII tubulin and GAP43 in the mouse ICBNs and ICNTs visualized using 3D confocal imaging as a function of the time mice are euthanized. Six corneas from three male and three female mice euthanized at 5:30 AM (ZT 22.5), 8:30 AM (ZT 1.5), 5:30 PM (ZT 10.5), and 8:30 PM (ZT 13.5) were processed to visualize βIII tubulin and GAP43. Representative confocal images from a male cornea were acquired at the basal aspect of the corneal epithelium to show the ICBNs, at the middle of the corneal epithelium showing ICNTs perpendicular to the basal surface that appear as puncta, and the apical aspect of the cornea where many of the ICNTs turn and extend parallel to the ocular surface forming parallel ICNTs (pICNTs). The regions indicated by the asterisks in the apical images were magnified × 3 and are presented at the far right to highlight the pICNTs and their loss between ZT 22.5 and ZT 1.5. The localization of GAP43 and βIII tubulin varies within the axons; these data are quantified in Figure 3 . Scale bar: 25 µm.
Figure Legend Snippet: Localization of axonal proteins βIII tubulin and GAP43 in the mouse ICBNs and ICNTs visualized using 3D confocal imaging as a function of the time mice are euthanized. Six corneas from three male and three female mice euthanized at 5:30 AM (ZT 22.5), 8:30 AM (ZT 1.5), 5:30 PM (ZT 10.5), and 8:30 PM (ZT 13.5) were processed to visualize βIII tubulin and GAP43. Representative confocal images from a male cornea were acquired at the basal aspect of the corneal epithelium to show the ICBNs, at the middle of the corneal epithelium showing ICNTs perpendicular to the basal surface that appear as puncta, and the apical aspect of the cornea where many of the ICNTs turn and extend parallel to the ocular surface forming parallel ICNTs (pICNTs). The regions indicated by the asterisks in the apical images were magnified × 3 and are presented at the far right to highlight the pICNTs and their loss between ZT 22.5 and ZT 1.5. The localization of GAP43 and βIII tubulin varies within the axons; these data are quantified in Figure 3 . Scale bar: 25 µm.

Techniques Used: Imaging, Mouse Assay

25) Product Images from "Evidence for Paracrine Protective Role of Exogenous αA-Crystallin in Retinal Ganglion Cells"

Article Title: Evidence for Paracrine Protective Role of Exogenous αA-Crystallin in Retinal Ganglion Cells

Journal: eNeuro

doi: 10.1523/ENEURO.0045-22.2022

Exogenous αA-crystallin protects primary mice retinal ganglion cells under stress. ( A ) Seven days post seeding, the RGCs show prominent neural processes. ( B ) Immunofluorescence analyses highlight prominent staining for neuron-specific βIII-tubulin (green), RBPMS (red) and Hoechst (blue). ( C ) Statistical analyses of RGC viability following exposure to stress. Percentage of apoptotic cells (TUNEL positive) in all RGCs (RBPMS positive) were analyzed. Data are represented as mean ± S.D. Statistical comparisons between groups were calculated by One-Way ANOVA followed by Tukey post-hoc tests. (** p ≤ 0.01), (*** p ≤ 0.001), (**** p ≤ 0.0001), significantly different from respective EV-transfected cells. ( D ) Vehicle control (VC), Recombinant wild type αA-crystallin (WT), αA-crystallin phosphomimetic (T148D), and the non-phosphorylatable form of αA-crystallin (T148A) were supplemented to RGCs at a 500 ng/ml concentration and incubated for 8 hours with 25 m m D-glucose (HG) and 100 ng/ml TNFα for 8 hours. Cells incubated with 5 m m glucose (NG) served as an experimental control. RGC survival under stress was assessed by TUNEL staining (green), and cells were later stained for RBPMS (red).
Figure Legend Snippet: Exogenous αA-crystallin protects primary mice retinal ganglion cells under stress. ( A ) Seven days post seeding, the RGCs show prominent neural processes. ( B ) Immunofluorescence analyses highlight prominent staining for neuron-specific βIII-tubulin (green), RBPMS (red) and Hoechst (blue). ( C ) Statistical analyses of RGC viability following exposure to stress. Percentage of apoptotic cells (TUNEL positive) in all RGCs (RBPMS positive) were analyzed. Data are represented as mean ± S.D. Statistical comparisons between groups were calculated by One-Way ANOVA followed by Tukey post-hoc tests. (** p ≤ 0.01), (*** p ≤ 0.001), (**** p ≤ 0.0001), significantly different from respective EV-transfected cells. ( D ) Vehicle control (VC), Recombinant wild type αA-crystallin (WT), αA-crystallin phosphomimetic (T148D), and the non-phosphorylatable form of αA-crystallin (T148A) were supplemented to RGCs at a 500 ng/ml concentration and incubated for 8 hours with 25 m m D-glucose (HG) and 100 ng/ml TNFα for 8 hours. Cells incubated with 5 m m glucose (NG) served as an experimental control. RGC survival under stress was assessed by TUNEL staining (green), and cells were later stained for RBPMS (red).

Techniques Used: Mouse Assay, Immunofluorescence, Staining, TUNEL Assay, Transfection, Recombinant, Concentration Assay, Incubation

26) Product Images from "Protective Effect of Human-Neural-Crest-Derived Nasal Turbinate Stem Cells against Amyloid-β Neurotoxicity through Inhibition of Osteopontin in a Human Cerebral Organoid Model of Alzheimer’s Disease"

Article Title: Protective Effect of Human-Neural-Crest-Derived Nasal Turbinate Stem Cells against Amyloid-β Neurotoxicity through Inhibition of Osteopontin in a Human Cerebral Organoid Model of Alzheimer’s Disease

Journal: Cells

doi: 10.3390/cells11061029

Histological analysis of hBOs cultured in the presence of Aβ 1–42 . ( A – E ) The hBOs were cultured in the absence or presence of 10 μM Aβ 1–42 for 72–80 h. Confocal microscopy images of hBOs after staining OCT-embedded sections with a TUNEL assay kit (red) or with antibodies against tubulin β-III tubulin, NeuN, and Nestin (green). Nuclei were labeled with DAPI (blue). Scale bars: 50 μm, 20 μm, or 200 μm. ( F ) Confocal microscopy images of hBOs cultured in the presence of 10 μM Aβ 1–42 after staining of OCT-embedded sections with an antibody against 6E10 to detect Aβ deposition (green). Nuclei were labeled with DAPI (blue). Scale bars: 200 μm or 50 μm. ( G ) Ki67, Tubulin β-III tubulin, Nestin, and NeuN mRNA levels were measured at 72–80 h after culture by quantitative real-time PCR in hBOs cultured with or without Aβ 1–42 . The GAPDH gene was used as a control. The values shown are the mean (SD). The significance of differences between two different samples was determined with Student’s t -test. * p
Figure Legend Snippet: Histological analysis of hBOs cultured in the presence of Aβ 1–42 . ( A – E ) The hBOs were cultured in the absence or presence of 10 μM Aβ 1–42 for 72–80 h. Confocal microscopy images of hBOs after staining OCT-embedded sections with a TUNEL assay kit (red) or with antibodies against tubulin β-III tubulin, NeuN, and Nestin (green). Nuclei were labeled with DAPI (blue). Scale bars: 50 μm, 20 μm, or 200 μm. ( F ) Confocal microscopy images of hBOs cultured in the presence of 10 μM Aβ 1–42 after staining of OCT-embedded sections with an antibody against 6E10 to detect Aβ deposition (green). Nuclei were labeled with DAPI (blue). Scale bars: 200 μm or 50 μm. ( G ) Ki67, Tubulin β-III tubulin, Nestin, and NeuN mRNA levels were measured at 72–80 h after culture by quantitative real-time PCR in hBOs cultured with or without Aβ 1–42 . The GAPDH gene was used as a control. The values shown are the mean (SD). The significance of differences between two different samples was determined with Student’s t -test. * p

Techniques Used: Cell Culture, Confocal Microscopy, Staining, TUNEL Assay, Labeling, Real-time Polymerase Chain Reaction

Characterization of hBOs in culture. ( A ) Confocal microscopy images of hBOs stained with antibodies against Nestin, GFAP, Tubulin β-III, and NeuN at day 60 in differentiation medium. Scale bars: 50 μm or 20 μm. ( B ) Confocal fluorescent image overlaid on the DIC image of hBO. Scale bar: 500 μm. ( C , D ) TEM images of cortical organoids showing synaptic clefts (head arrows) and synaptic vesicles (asterisks). Scale bar: 500 nm. All images are representative of two or three independent experiments.
Figure Legend Snippet: Characterization of hBOs in culture. ( A ) Confocal microscopy images of hBOs stained with antibodies against Nestin, GFAP, Tubulin β-III, and NeuN at day 60 in differentiation medium. Scale bars: 50 μm or 20 μm. ( B ) Confocal fluorescent image overlaid on the DIC image of hBO. Scale bar: 500 μm. ( C , D ) TEM images of cortical organoids showing synaptic clefts (head arrows) and synaptic vesicles (asterisks). Scale bar: 500 nm. All images are representative of two or three independent experiments.

Techniques Used: Confocal Microscopy, Staining, Transmission Electron Microscopy

27) Product Images from "Direct Reprogramming of Human Neurons Identifies MARCKSL1 as a Pathogenic Mediator of Valproic Acid-Induced Teratogenicity"

Article Title: Direct Reprogramming of Human Neurons Identifies MARCKSL1 as a Pathogenic Mediator of Valproic Acid-Induced Teratogenicity

Journal: Cell stem cell

doi: 10.1016/j.stem.2019.04.021

Directly reprogrammed human neurons rapidly acquire neuronal identity, but undergo distinct developmental stages and gradual maturation in vitro . A-B. Neurogenesis was induced in H1-ES cells by Ngn2 expression. Neurons were co-cultured with glia and analyzed (arrowheads) at different time-points ( A ). In some experiments, cells were additionally infected with a lentivirus expressing EGFP. Example images of day 4 human neurons derived from an ES-cell colony ( B ). C. Sample images (left) of ES cells and day 4 neurons (EGFP expressing) stained with DAPI and antibody for Nanog. Summary graph (right) of average intensity for Nanog signal normalized by DAPI-positive nuclear area. D. Representative images of ES cells and human neurons at post-induction day 4, day 7, day 14, day 21, day 30, and day 45 (vertical columns), as stained with DAPI and immunolabeled for indicated markers (horizontal rows). White arrowheads = human cells (ES cells or neurons), yellow arrowheads = glia, and cyan arrowheads = dividing ES cells. E-M. Quantifications of signal-intensities for different markers expressed in ES cells and at different time-points of neuronal maturation in vitro . E , Sox2 normalized to nuclear area; F , Ki-67 normalized to nucleus count; G-I , Nestin ( G ), Dcx ( H ) and Tuj1 ( I ) normalized to total surface area; J , NeuN normalized to nuclear area; K , Map2 normalized to neurite area; L-M , Synapsin normalized to threshold-adjusted MAP2 area ( L , summary graph; M , sample images). N. Example traces (top) of APs produced by a 90 pA step-current injection (protocol is shown on top, V hold = −60 mV) at day 4, day 7, day 14, day 21, day 30, and day 45; summary graphs (bottom) of the percentage of cells capable of firing APs (left) and the total number of APs (right). O. Summary graphs of C m (left), R m (middle), or V m (right) at different time-points of neuronal maturation. P. Sample traces of evoked AMPAR-EPSCs at different time-points (left); summary graphs for the percentage of cells with AMPAR-EPSCs (middle), and peak-amplitude of AMPAR-EPSCs (right). All summary data are presented as means ± SEM, with total number of cells and dendritic sections analyzed (for immunostaining) or cells patched (for electrophysiology) / number of independent experiments.
Figure Legend Snippet: Directly reprogrammed human neurons rapidly acquire neuronal identity, but undergo distinct developmental stages and gradual maturation in vitro . A-B. Neurogenesis was induced in H1-ES cells by Ngn2 expression. Neurons were co-cultured with glia and analyzed (arrowheads) at different time-points ( A ). In some experiments, cells were additionally infected with a lentivirus expressing EGFP. Example images of day 4 human neurons derived from an ES-cell colony ( B ). C. Sample images (left) of ES cells and day 4 neurons (EGFP expressing) stained with DAPI and antibody for Nanog. Summary graph (right) of average intensity for Nanog signal normalized by DAPI-positive nuclear area. D. Representative images of ES cells and human neurons at post-induction day 4, day 7, day 14, day 21, day 30, and day 45 (vertical columns), as stained with DAPI and immunolabeled for indicated markers (horizontal rows). White arrowheads = human cells (ES cells or neurons), yellow arrowheads = glia, and cyan arrowheads = dividing ES cells. E-M. Quantifications of signal-intensities for different markers expressed in ES cells and at different time-points of neuronal maturation in vitro . E , Sox2 normalized to nuclear area; F , Ki-67 normalized to nucleus count; G-I , Nestin ( G ), Dcx ( H ) and Tuj1 ( I ) normalized to total surface area; J , NeuN normalized to nuclear area; K , Map2 normalized to neurite area; L-M , Synapsin normalized to threshold-adjusted MAP2 area ( L , summary graph; M , sample images). N. Example traces (top) of APs produced by a 90 pA step-current injection (protocol is shown on top, V hold = −60 mV) at day 4, day 7, day 14, day 21, day 30, and day 45; summary graphs (bottom) of the percentage of cells capable of firing APs (left) and the total number of APs (right). O. Summary graphs of C m (left), R m (middle), or V m (right) at different time-points of neuronal maturation. P. Sample traces of evoked AMPAR-EPSCs at different time-points (left); summary graphs for the percentage of cells with AMPAR-EPSCs (middle), and peak-amplitude of AMPAR-EPSCs (right). All summary data are presented as means ± SEM, with total number of cells and dendritic sections analyzed (for immunostaining) or cells patched (for electrophysiology) / number of independent experiments.

Techniques Used: In Vitro, Expressing, Cell Culture, Infection, Derivative Assay, Staining, Immunolabeling, Produced, Injection, Immunostaining

VPA impairs dendritic morphogenesis and synaptic maturation of developing neurons. A. Experimental time-course: the Ngn2-induced human neurons (co-expressing EGFP) were treated with VPA or equal volume of dH 2 O (control) at day 1 (blue arrowhead) for the next 72 hr, and subsequently analyzed at different time-points (black arrowheads). B. Representative images of VPA-treated day 4 neurons immunostained for Tuj1 (left); summary graph of total neurite length normalized by the count of DAPI-stained nucleus (right). C-D. Sample images ( C ) of EGFP-expressing neurons that were exposed to VPA of indicated concentrations; summary graphs ( D ) of neurite length (top) and number of branches (bottom). E-F. Sample images ( E , gray arrowhead = conventional neurites, white arrowhead = filopodia-like extensions); summary graphs ( F ) of soma size (top left), percentage of cells with filopodia (top right, connected circles are values from individual batches), filopodia number (bottom left) and length (bottom right). G-H. Kinetics of neurite outgrowth in cells that were examined every fifth day after VPA exposure ( G , example images, arrowheads = cell-bodies; H , summary graph of neurite length). Single neuron labeling was achieved by sparse infection with a low titer lentivirus expressing EGFP. I-J. Representative images ( H ) of control vs. VPA-treated cells, double-labeled for dendritic MAP2 and synaptic Synapsin proteins (boxes expanded on right); summary graphs ( I ) of dendritic (top, left to right: dendrite length, branch number, and primary dendrites) and synaptic (bottom, left to right: computed total number of synapses, synapse density and size) parameters. K. Summary graphs of C m (left), R m (middle), or V m (right), for control vs. VPA-exposed neurons. L. Example traces (left) of APs produced by step-currents (protocol is shown on top, V hold = −60 mV); summary plot (right) of AP numbers as a function of injected current-amplitude. M. Superimposed sample traces (left) of AMPAR-mediated evoked EPSCs; summary graphs (right) of the EPSC amplitude, and CV of EPSCs as an indirect measure of presynaptic release probability. All data are means ± SEM, with number of frames analyzed (imaging), or cells patched (electrophysiology) / number of batches. Statistical significance was weighed by two-way ANOVA (for L ) or two-tailed, unpaired, Student’s t-test (all bar-graphs), with *** P
Figure Legend Snippet: VPA impairs dendritic morphogenesis and synaptic maturation of developing neurons. A. Experimental time-course: the Ngn2-induced human neurons (co-expressing EGFP) were treated with VPA or equal volume of dH 2 O (control) at day 1 (blue arrowhead) for the next 72 hr, and subsequently analyzed at different time-points (black arrowheads). B. Representative images of VPA-treated day 4 neurons immunostained for Tuj1 (left); summary graph of total neurite length normalized by the count of DAPI-stained nucleus (right). C-D. Sample images ( C ) of EGFP-expressing neurons that were exposed to VPA of indicated concentrations; summary graphs ( D ) of neurite length (top) and number of branches (bottom). E-F. Sample images ( E , gray arrowhead = conventional neurites, white arrowhead = filopodia-like extensions); summary graphs ( F ) of soma size (top left), percentage of cells with filopodia (top right, connected circles are values from individual batches), filopodia number (bottom left) and length (bottom right). G-H. Kinetics of neurite outgrowth in cells that were examined every fifth day after VPA exposure ( G , example images, arrowheads = cell-bodies; H , summary graph of neurite length). Single neuron labeling was achieved by sparse infection with a low titer lentivirus expressing EGFP. I-J. Representative images ( H ) of control vs. VPA-treated cells, double-labeled for dendritic MAP2 and synaptic Synapsin proteins (boxes expanded on right); summary graphs ( I ) of dendritic (top, left to right: dendrite length, branch number, and primary dendrites) and synaptic (bottom, left to right: computed total number of synapses, synapse density and size) parameters. K. Summary graphs of C m (left), R m (middle), or V m (right), for control vs. VPA-exposed neurons. L. Example traces (left) of APs produced by step-currents (protocol is shown on top, V hold = −60 mV); summary plot (right) of AP numbers as a function of injected current-amplitude. M. Superimposed sample traces (left) of AMPAR-mediated evoked EPSCs; summary graphs (right) of the EPSC amplitude, and CV of EPSCs as an indirect measure of presynaptic release probability. All data are means ± SEM, with number of frames analyzed (imaging), or cells patched (electrophysiology) / number of batches. Statistical significance was weighed by two-way ANOVA (for L ) or two-tailed, unpaired, Student’s t-test (all bar-graphs), with *** P

Techniques Used: Expressing, Staining, Labeling, Infection, Produced, Injection, Imaging, Two Tailed Test

28) Product Images from "Human Deep Cortical Neurons Promote Regeneration and Recovery After Cervical Spinal Cord Injury"

Article Title: Human Deep Cortical Neurons Promote Regeneration and Recovery After Cervical Spinal Cord Injury

Journal: bioRxiv

doi: 10.1101/2021.08.11.455948

Human iPSC-DCNs maintain their deep cortical identity in vivo and reduce cavitation and inflammation. A. Grafted GFP + iPSC-DCNs are TUJ1 + , human NCAM + , NFH + ; Ctip2 + , TBR1 + , Homer + and VGlut 2 + . Low numbers of GFP + iPSC-DCNs are Stathmin + , Nestin + , SC123 + , but not Satb2 + or Olig2 + . Scale bars = 50 µm. B. Shaded areas show cavitation from C4-C8 in cords of transplanted and control rats. C. Lesion cavity volume results are represented as box and whisker plots to visualize distribution; (red = no cells, blue = cells). Tukey HSD Test; * P ≤ 0.05 , ** P ≤ 0.01 . N=11, 3, 7, and 16 respectively. D. GFP + human iPSC-DCNs ( D’ ) fill the lesion cavity (dotted outline) ( D ). Scale bars = 500 µm. E-F. iPSC-DCNs (n=8) reduce GFAP + astrogliosis compared to injury controls (n=6) at rostral, lesion, and caudal segments. Fluorescence intensity averages are represented as columns ± SEM. Tukey HSD Test; * P ≤ 0.05 . Scale bars = 500 µm. G-H. After 12 weeks, iPSC-DCN transplantation (n=8) reduces Iba1 + microglia/macrophage presence compared to injury controls (n=6). Fluorescence intensity averages are represented as points ± SEM. Welch’s T-Test; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Scale bars = 500 µm.
Figure Legend Snippet: Human iPSC-DCNs maintain their deep cortical identity in vivo and reduce cavitation and inflammation. A. Grafted GFP + iPSC-DCNs are TUJ1 + , human NCAM + , NFH + ; Ctip2 + , TBR1 + , Homer + and VGlut 2 + . Low numbers of GFP + iPSC-DCNs are Stathmin + , Nestin + , SC123 + , but not Satb2 + or Olig2 + . Scale bars = 50 µm. B. Shaded areas show cavitation from C4-C8 in cords of transplanted and control rats. C. Lesion cavity volume results are represented as box and whisker plots to visualize distribution; (red = no cells, blue = cells). Tukey HSD Test; * P ≤ 0.05 , ** P ≤ 0.01 . N=11, 3, 7, and 16 respectively. D. GFP + human iPSC-DCNs ( D’ ) fill the lesion cavity (dotted outline) ( D ). Scale bars = 500 µm. E-F. iPSC-DCNs (n=8) reduce GFAP + astrogliosis compared to injury controls (n=6) at rostral, lesion, and caudal segments. Fluorescence intensity averages are represented as columns ± SEM. Tukey HSD Test; * P ≤ 0.05 . Scale bars = 500 µm. G-H. After 12 weeks, iPSC-DCN transplantation (n=8) reduces Iba1 + microglia/macrophage presence compared to injury controls (n=6). Fluorescence intensity averages are represented as points ± SEM. Welch’s T-Test; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Scale bars = 500 µm.

Techniques Used: In Vivo, Whisker Assay, Fluorescence, Transplantation Assay

29) Product Images from "Eosinophils regulate intra-adipose axonal plasticity"

Article Title: Eosinophils regulate intra-adipose axonal plasticity

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.2112281119

The sympathetic nerves are in proximity with IL-33 and eosinophils in mouse and human adipose tissues. ( A and B ) The mouse iWAT were processed for the whole-mount immunostaining and volume fluorescence imaging. The 3D-projection images of TH and IL-33 adjacent to blood vessel ( A , Top ) and in the parenchyma ( A , Bottom ) were shown with a depth of 500 μm. The distance of IL-33 from nerve was plotted ( B ). n = 7 mice. ( C ) Human omental adipose tissues were processed by cryosections and immunostaining. The costained images of Tuj1 and IL-33, Tuj1 and Siglec-8, or Tuj1 and CD45 were shown.
Figure Legend Snippet: The sympathetic nerves are in proximity with IL-33 and eosinophils in mouse and human adipose tissues. ( A and B ) The mouse iWAT were processed for the whole-mount immunostaining and volume fluorescence imaging. The 3D-projection images of TH and IL-33 adjacent to blood vessel ( A , Top ) and in the parenchyma ( A , Bottom ) were shown with a depth of 500 μm. The distance of IL-33 from nerve was plotted ( B ). n = 7 mice. ( C ) Human omental adipose tissues were processed by cryosections and immunostaining. The costained images of Tuj1 and IL-33, Tuj1 and Siglec-8, or Tuj1 and CD45 were shown.

Techniques Used: Immunostaining, Fluorescence, Imaging, Mouse Assay

30) Product Images from "The Class I E3 Ubiquitin Ligase TRIM67 Modulates Brain Development and Behavior"

Article Title: The Class I E3 Ubiquitin Ligase TRIM67 Modulates Brain Development and Behavior

Journal: bioRxiv

doi: 10.1101/241331

Generation of Trim67 -/- mouse and TRIM67 brain localization. A Diagram of targeting strategy for knockout of the Trim67 gene showing Cre-lox mediated excision of exon 1. This excision leads to the next 16 intronic and 4 exonic ATG codons being out of frame. LoxP sites are shown in yellow, FLIP sites in orange, and neomycin resistance gene (NeoR) in purple. B Agarose gel separation of genotyping PCR products, demonstrating deletion of both copies of the first exon of Trim67 ( -/- ) or one copy in a heterozygote (-/+). Diagrams show PCR products from Trim67 +/+ and Trim67 -/- DNA. C Mendelian ratio of Trim67 allele inheritance in 48 litters of heterozygous knockout crosses, showing approximately expected rates of inheritance of each allele (p = 0.117, chi-square test). D Western blot of whole-brain or two day in vitro dissociated cortical neuron lysate from Trim67 +/+ and Trim67 -/- E15.5 embryos probed for TRIM67 and βIII-tubulin as a loading control. E Fluorescent micrographs of E15.5 and P140 brain sections shows expression and loss of TRIM67 (green) in Trim67 +/+ and Trim67 -/- mice, respectively. Sections are counterstained for β-III-tubulin (magenta) and with DAPI (blue). Scale bar 50μm. F Western blot detects TRIM67 expression in various adult and embryonic brain tissues, but not outside the nervous system. GAPDH is a loading control.
Figure Legend Snippet: Generation of Trim67 -/- mouse and TRIM67 brain localization. A Diagram of targeting strategy for knockout of the Trim67 gene showing Cre-lox mediated excision of exon 1. This excision leads to the next 16 intronic and 4 exonic ATG codons being out of frame. LoxP sites are shown in yellow, FLIP sites in orange, and neomycin resistance gene (NeoR) in purple. B Agarose gel separation of genotyping PCR products, demonstrating deletion of both copies of the first exon of Trim67 ( -/- ) or one copy in a heterozygote (-/+). Diagrams show PCR products from Trim67 +/+ and Trim67 -/- DNA. C Mendelian ratio of Trim67 allele inheritance in 48 litters of heterozygous knockout crosses, showing approximately expected rates of inheritance of each allele (p = 0.117, chi-square test). D Western blot of whole-brain or two day in vitro dissociated cortical neuron lysate from Trim67 +/+ and Trim67 -/- E15.5 embryos probed for TRIM67 and βIII-tubulin as a loading control. E Fluorescent micrographs of E15.5 and P140 brain sections shows expression and loss of TRIM67 (green) in Trim67 +/+ and Trim67 -/- mice, respectively. Sections are counterstained for β-III-tubulin (magenta) and with DAPI (blue). Scale bar 50μm. F Western blot detects TRIM67 expression in various adult and embryonic brain tissues, but not outside the nervous system. GAPDH is a loading control.

Techniques Used: Knock-Out, Agarose Gel Electrophoresis, Genotyping Assay, Polymerase Chain Reaction, Western Blot, In Vitro, Expressing, Mouse Assay

TRIM67 is expressed in multiple murine brain regions. Sagittal sections of E15.5 ( A-C ) and P140 ( D-F ) brains stained for TRIM67 (green), β-III-tubulin (red), and nuclei (DAPI, blue). A TRIM67 is expressed in cell bodies in the developing hippocampus at E15.5 (scale bar = 50 μm). B,C TRIM67 expression is evident in the peduncular hypothalamus (B, C5), diencephalon (C1), and reticular complex (C2), but not in the prethalamic eminence (C3), zona incerta complex (C4) or subpallium (C6) (scale bars B,C = 200μm). D-F In the adult brain, TRIM67 expression is evident in fiber bundles of the corticospinal tract passing through the caudate putamen (arrowheads, D) and scattered cells in the lateral hypothalamic area (arrowheads, E ), but not in the hippocampus ( F ). (scale bar = 100μm).
Figure Legend Snippet: TRIM67 is expressed in multiple murine brain regions. Sagittal sections of E15.5 ( A-C ) and P140 ( D-F ) brains stained for TRIM67 (green), β-III-tubulin (red), and nuclei (DAPI, blue). A TRIM67 is expressed in cell bodies in the developing hippocampus at E15.5 (scale bar = 50 μm). B,C TRIM67 expression is evident in the peduncular hypothalamus (B, C5), diencephalon (C1), and reticular complex (C2), but not in the prethalamic eminence (C3), zona incerta complex (C4) or subpallium (C6) (scale bars B,C = 200μm). D-F In the adult brain, TRIM67 expression is evident in fiber bundles of the corticospinal tract passing through the caudate putamen (arrowheads, D) and scattered cells in the lateral hypothalamic area (arrowheads, E ), but not in the hippocampus ( F ). (scale bar = 100μm).

Techniques Used: Staining, Expressing

31) Product Images from "hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function"

Article Title: hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

Journal: bioRxiv

doi: 10.1101/2022.01.04.474746

PDGFR inhibition promotes enteric NO neuron induction A) Schematic representation of a high-throughput pharmacological screening to identify compounds that enrich NO neurons in hESC-derived 2D ENS cultures. B) Combined protein target analysis for the HTS top 12 hits showing shared protein classes between structurally similar hits. C) Effect of PP121 treatment window on NOS1::GFP induction efficiency. D) Immunofluorescence staining of NOS1 and neuronal TUBB3 in stage 1 enteric ganglioids treated with or without PP121 between days 15 and 20. E) Split UMAP of cell types present in stage 1 control (top) and PP121 treated (bottom) enteric ganglioid cultures. F) Dot plot of the average module scores of control only enteric ganglioid subtype transcriptional signatures in PP121 treated ganglioidl subtypes. G) Split UMAP of neuronal subtypes present in stage 1 control (top) and PP121 treated (bottom) enteric ganglioid cultures. H) Dot plot of the average module scores of control only neuronal subtype transcriptional signatures in PP121 treated ganglioid neuronal subtypes. I) Distribution of NO neuron subtypes in control versus PP121 treated stage 1 enteric ganglioid cultures. J) Split UMAP of subclustered NO subtypes present in stage 1 control (top) and PP121 treated (bottom) enteric ganglioid cultures. K) Dot plot of the average module scores of control only NO neuron subtype transcriptional signatures in PP121 treated ganglioid NO neuron subtypes. L) Feature plot showing the expression of ERBBs, PDGFRs and VEGFRs in D15 subclustered enteric crestospheres. M) Schematic of receptor tyrosine kinase (RTK) natural agonists and selected pharmacological antagonists including NO neuron enriching top hit PP121. N) Effect of RTK ligand treatment on stage 1 enteric ganglioid NO neuron induction. O and P ) Effect of knocking out PDGFRA ( O ) and PDGFRB ( P ) in D15 enteric crestospheres on stage 1 enteric ganglioid NO neuron enrichment as measured by flow cytometry.
Figure Legend Snippet: PDGFR inhibition promotes enteric NO neuron induction A) Schematic representation of a high-throughput pharmacological screening to identify compounds that enrich NO neurons in hESC-derived 2D ENS cultures. B) Combined protein target analysis for the HTS top 12 hits showing shared protein classes between structurally similar hits. C) Effect of PP121 treatment window on NOS1::GFP induction efficiency. D) Immunofluorescence staining of NOS1 and neuronal TUBB3 in stage 1 enteric ganglioids treated with or without PP121 between days 15 and 20. E) Split UMAP of cell types present in stage 1 control (top) and PP121 treated (bottom) enteric ganglioid cultures. F) Dot plot of the average module scores of control only enteric ganglioid subtype transcriptional signatures in PP121 treated ganglioidl subtypes. G) Split UMAP of neuronal subtypes present in stage 1 control (top) and PP121 treated (bottom) enteric ganglioid cultures. H) Dot plot of the average module scores of control only neuronal subtype transcriptional signatures in PP121 treated ganglioid neuronal subtypes. I) Distribution of NO neuron subtypes in control versus PP121 treated stage 1 enteric ganglioid cultures. J) Split UMAP of subclustered NO subtypes present in stage 1 control (top) and PP121 treated (bottom) enteric ganglioid cultures. K) Dot plot of the average module scores of control only NO neuron subtype transcriptional signatures in PP121 treated ganglioid NO neuron subtypes. L) Feature plot showing the expression of ERBBs, PDGFRs and VEGFRs in D15 subclustered enteric crestospheres. M) Schematic of receptor tyrosine kinase (RTK) natural agonists and selected pharmacological antagonists including NO neuron enriching top hit PP121. N) Effect of RTK ligand treatment on stage 1 enteric ganglioid NO neuron induction. O and P ) Effect of knocking out PDGFRA ( O ) and PDGFRB ( P ) in D15 enteric crestospheres on stage 1 enteric ganglioid NO neuron enrichment as measured by flow cytometry.

Techniques Used: Inhibition, High Throughput Screening Assay, Derivative Assay, Immunofluorescence, Staining, Expressing, Flow Cytometry

hPSC-derived enteric ganglioids model human ENS neurochemical diversity A) snRNA-seq UMAP of neuronal subtypes present in stage 1 enteric ganglioids. B) snRNA-seq UMAP of neuronal subtypes present in stage 2 enteric ganglioids. C) Projection of stage 2 neuronal subtypes (right) onto the SWNE of stage 1 enteric ganglioid neurons with overlayed projection rate-limiting neurotransmitter synthesis enzymes. D) Dot plot of the average module scores of stage 1 (bottom) and stage 2 (top) ganglioid cell type transcriptional signatures adult human colon cell types. E) Dot plot of the average module scores of stage 1 (bottom) and stage 2 (top) ganglioid neuronal subtype transcriptional signatures adult human enteric neuron subtypes. F) Immunofluorescence analysis for expression of ENS cell-type markers (serotonin, CHAT, GABA and NOS1) in stage 1 enteric ganglioids. G) Quantification of flow cytometry analysis for the expression of neuronal subtype markers serotonin, CHAT, GABA and NOS1 in stage 1 2D ENS cultures (left) and 3D enteric ganglioids (right). H) Flow cytometry validation of stage 1 EN 8 surface markers CCR6 (left) and GYPB (right) co-labeling with neurochemical markers showing enrichment of neurochemical identities of marker positive populations normalized to baseline neurochemical population levels. I) Overall percentage of neurotransmitter synthesizing neurons in stage 1 and 2 enteric ganglioids compared to mouse and human primary enteric neurons. J) Schematic of mono- and multi-neurotransmitter synthesis in enteric neurons. K) Percentage of neurons showing mono-and multi-neurotransmitter profiles in stage 1 and 2 enteric ganglioid neurons compared to mouse and human primary enteric neurons. L) Immunostaining of prima ry human colon with antibodies against NOS1, GABA and TUBB3 (top), and CHAT, GABA and TUBB3 (bottom). White dash line indicates the border of TUBB3 + ganglia. White arrows indicate colocalization. M) Percentage of mono-neurotransmitter (top) and bi-neurotransmitter (bottom) producing enteric neurons in stage 1, 2 enteric ganglioids and primary datasets.
Figure Legend Snippet: hPSC-derived enteric ganglioids model human ENS neurochemical diversity A) snRNA-seq UMAP of neuronal subtypes present in stage 1 enteric ganglioids. B) snRNA-seq UMAP of neuronal subtypes present in stage 2 enteric ganglioids. C) Projection of stage 2 neuronal subtypes (right) onto the SWNE of stage 1 enteric ganglioid neurons with overlayed projection rate-limiting neurotransmitter synthesis enzymes. D) Dot plot of the average module scores of stage 1 (bottom) and stage 2 (top) ganglioid cell type transcriptional signatures adult human colon cell types. E) Dot plot of the average module scores of stage 1 (bottom) and stage 2 (top) ganglioid neuronal subtype transcriptional signatures adult human enteric neuron subtypes. F) Immunofluorescence analysis for expression of ENS cell-type markers (serotonin, CHAT, GABA and NOS1) in stage 1 enteric ganglioids. G) Quantification of flow cytometry analysis for the expression of neuronal subtype markers serotonin, CHAT, GABA and NOS1 in stage 1 2D ENS cultures (left) and 3D enteric ganglioids (right). H) Flow cytometry validation of stage 1 EN 8 surface markers CCR6 (left) and GYPB (right) co-labeling with neurochemical markers showing enrichment of neurochemical identities of marker positive populations normalized to baseline neurochemical population levels. I) Overall percentage of neurotransmitter synthesizing neurons in stage 1 and 2 enteric ganglioids compared to mouse and human primary enteric neurons. J) Schematic of mono- and multi-neurotransmitter synthesis in enteric neurons. K) Percentage of neurons showing mono-and multi-neurotransmitter profiles in stage 1 and 2 enteric ganglioid neurons compared to mouse and human primary enteric neurons. L) Immunostaining of prima ry human colon with antibodies against NOS1, GABA and TUBB3 (top), and CHAT, GABA and TUBB3 (bottom). White dash line indicates the border of TUBB3 + ganglia. White arrows indicate colocalization. M) Percentage of mono-neurotransmitter (top) and bi-neurotransmitter (bottom) producing enteric neurons in stage 1, 2 enteric ganglioids and primary datasets.

Techniques Used: Derivative Assay, Immunofluorescence, Expressing, Flow Cytometry, Labeling, Marker, Immunostaining

Human surface marker antibody screening identifies NO neuron specific surface markers A) Schematic representation of using enriched enteric NO neuron cultures to identify their specific surface markers by a flow cytometry-based high-throughput antibody screening. B) Surface markers that were expressed in at least 50% of NOS1 + cells, i.e. showing > 50% sensitivity (top) were filtered based on their specificity for NOS1 + cells (at least 70% of all stained cells being NOS1 + , i.e. > 70% specificity, middle). C) Surface marker screening top hits with the highest specificity as well as sensitivity for identifying enteric NO neurons. D) Violin plot stack showing the expression (left) and dot plot showing the average scaled expression (right) of surface marker screen hits in control and PP121 treated NO neuron subtypes versus other neurons. E) Violin plot stack showing the expression of surface marker screen hits in adult human NO neuron subtypes versus other neurons. F) Immunofluorescence analysis for expression of neuronal marker TUBB3, NOS1 and CD47 in adult human primary colon tissue. A myenteric plexus ganglion is shown. Arrows show colocalization of CD47 and NOS1. G) Surface markers that stained at least 70% of total cells as potential pan ENS cell markers. H) CD24 expression in stage 1 enteric ganglioid clusters in PP121 treated, and untreated samples. I) Representative immunofluorescence analysis of CD24 and neuronal marker TUBB3 expression in a human primary colonic section, showing a myenteric plexus ganglion.
Figure Legend Snippet: Human surface marker antibody screening identifies NO neuron specific surface markers A) Schematic representation of using enriched enteric NO neuron cultures to identify their specific surface markers by a flow cytometry-based high-throughput antibody screening. B) Surface markers that were expressed in at least 50% of NOS1 + cells, i.e. showing > 50% sensitivity (top) were filtered based on their specificity for NOS1 + cells (at least 70% of all stained cells being NOS1 + , i.e. > 70% specificity, middle). C) Surface marker screening top hits with the highest specificity as well as sensitivity for identifying enteric NO neurons. D) Violin plot stack showing the expression (left) and dot plot showing the average scaled expression (right) of surface marker screen hits in control and PP121 treated NO neuron subtypes versus other neurons. E) Violin plot stack showing the expression of surface marker screen hits in adult human NO neuron subtypes versus other neurons. F) Immunofluorescence analysis for expression of neuronal marker TUBB3, NOS1 and CD47 in adult human primary colon tissue. A myenteric plexus ganglion is shown. Arrows show colocalization of CD47 and NOS1. G) Surface markers that stained at least 70% of total cells as potential pan ENS cell markers. H) CD24 expression in stage 1 enteric ganglioid clusters in PP121 treated, and untreated samples. I) Representative immunofluorescence analysis of CD24 and neuronal marker TUBB3 expression in a human primary colonic section, showing a myenteric plexus ganglion.

Techniques Used: Marker, Flow Cytometry, High Throughput Screening Assay, Staining, Expressing, Immunofluorescence

hESC-derived enteric ganglioids engraft in adult mouse colon Immunohistochemical analysis of neuronal TUBB3, human cytoplasmic protein SC121, and NO neuron marker NOS1 in Nos1 -/- mouse colon (untreated with Cyclosporin A and non-operated, top) and 8 weeks post transplantation (bottom). Purple arrows point at NOS1 + cells.
Figure Legend Snippet: hESC-derived enteric ganglioids engraft in adult mouse colon Immunohistochemical analysis of neuronal TUBB3, human cytoplasmic protein SC121, and NO neuron marker NOS1 in Nos1 -/- mouse colon (untreated with Cyclosporin A and non-operated, top) and 8 weeks post transplantation (bottom). Purple arrows point at NOS1 + cells.

Techniques Used: Derivative Assay, Immunohistochemistry, Marker, Transplantation Assay

hPSC-derived enteric ganglioids model development, function and molecular diversity of human ENS A) Protocol schematic for in vitro differentiation and maturation of hPSCs into enteric neural crest and enteric crestospheres. B) scRNA-seq UMAP of cell types present in enteric neural crest cells (D10, top panel) and enteric crestosphere cells (D15, bottom panel) of the differentiation cultures depicted in ( A ). C) UMAP of enteric neural crest (D10, top) and enteric crestosphere (D15, bottom) subtypes in differentiation cultures. D) Violin plot stack showing the expression of canonical enteric neural crest markers in enteric neural crest (top) and enteric crestosphere (bottom) subtypes. E) Protocol schematic for in vitro differentiation and maturation of hPSC-derived enteric crestospheres into 2D ENS cultures and 3D ganglioids. F) snRNA-seq UMAP of cell types present in stage 1 enteric ganglioids. G) snRNA-seq UMAP of cell types present in stage 2 enteric ganglioids. H) Immunofluorescence analysis for expression of neuronal TUBB3 and glial GFAP in stage 1 and stage 2 enteric ganglioids. I) Immunofluorescence analysis for expression of neuronal activity marker cFOS in stage 1 and stage 2 enteric ganglioids. J) Flow cytometry quantification of neuronal activity marker cFOS in enteric ganglioids as they mature. K) Live fluorescence images of human hSYN-ChR2-EYFP in enteric ganglioids as they mature. L) Quantification of multi-electrode array (MEA) analysis of baseline and blue light-stimulated neuronal activity in stage 1 hSYN-ChR2-EYFP (left) and control (right) enteric ganglioids. M) Dot plot of the average module scores of stage 1 enteric ganglioid cell type transcriptional signatures in stage 2 enteric ganglioid cell types. N) Projection of stage 2 cell types (right) onto the SWNE of stage 1 enteric ganglioid cells with overlayed projection of stage 1 cell-type specific transcription factors from Figure S3 .
Figure Legend Snippet: hPSC-derived enteric ganglioids model development, function and molecular diversity of human ENS A) Protocol schematic for in vitro differentiation and maturation of hPSCs into enteric neural crest and enteric crestospheres. B) scRNA-seq UMAP of cell types present in enteric neural crest cells (D10, top panel) and enteric crestosphere cells (D15, bottom panel) of the differentiation cultures depicted in ( A ). C) UMAP of enteric neural crest (D10, top) and enteric crestosphere (D15, bottom) subtypes in differentiation cultures. D) Violin plot stack showing the expression of canonical enteric neural crest markers in enteric neural crest (top) and enteric crestosphere (bottom) subtypes. E) Protocol schematic for in vitro differentiation and maturation of hPSC-derived enteric crestospheres into 2D ENS cultures and 3D ganglioids. F) snRNA-seq UMAP of cell types present in stage 1 enteric ganglioids. G) snRNA-seq UMAP of cell types present in stage 2 enteric ganglioids. H) Immunofluorescence analysis for expression of neuronal TUBB3 and glial GFAP in stage 1 and stage 2 enteric ganglioids. I) Immunofluorescence analysis for expression of neuronal activity marker cFOS in stage 1 and stage 2 enteric ganglioids. J) Flow cytometry quantification of neuronal activity marker cFOS in enteric ganglioids as they mature. K) Live fluorescence images of human hSYN-ChR2-EYFP in enteric ganglioids as they mature. L) Quantification of multi-electrode array (MEA) analysis of baseline and blue light-stimulated neuronal activity in stage 1 hSYN-ChR2-EYFP (left) and control (right) enteric ganglioids. M) Dot plot of the average module scores of stage 1 enteric ganglioid cell type transcriptional signatures in stage 2 enteric ganglioid cell types. N) Projection of stage 2 cell types (right) onto the SWNE of stage 1 enteric ganglioid cells with overlayed projection of stage 1 cell-type specific transcription factors from Figure S3 .

Techniques Used: Derivative Assay, In Vitro, Expressing, Immunofluorescence, Activity Assay, Marker, Flow Cytometry, Fluorescence, Microelectrode Array

32) Product Images from "Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2"

Article Title: Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

Journal: EMBO Reports

doi: 10.15252/embr.202051913

Loss of Ndrg4 does not alter intestinal morphology or physiology Representative microscopic views of Ndrg4 +/+ and Ndrg4 −/− murine intestinal sections ( n = 4, 12 months of age) reveal an even distribution of alkaline phosphatase along the enterocyte brush border, and a similar number and distribution of Paneth cells (Lysozyme) in the small intestine; and neuroendocrine (chromogranin A), goblet (PAS+), and proliferating (Ki67) cells in the colon. Scale bars, 50 µm. Representative microscopic views of the myenteric plexus of Ndrg4 +/+ and Ndrg4 −/− murine colonic sections labeled with either TuJ1 and S100 (B, n = 3) or HuC/D (C, n = 6) did not reveal structural or organizational differences in the ganglionic network of Ndrg4 +/+ and Ndrg4 −/− mice. Scale bars, 100 µm. Quantification of the enteric neuronal cell number (HuC/D) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4 +/+ and Ndrg4 −/− mice ( n = 6). Gastrointestinal motility assays reveal a similar whole‐gut transit time (E; n = 12 versus 12), with a similar weight per stool and stool water content of the fecal pellets (F, G; n = 11 versus 10), a comparable small intestinal transit (H; n = 10 versus 12) and colonic propulsion time (I; n = 7 versus 6) in Ndrg4 −/− compared to Ndrg4 +/+ mice. Data information: All data are presented as mean ± SEM, with P ‐values determined using a two‐tailed, unpaired t ‐test.
Figure Legend Snippet: Loss of Ndrg4 does not alter intestinal morphology or physiology Representative microscopic views of Ndrg4 +/+ and Ndrg4 −/− murine intestinal sections ( n = 4, 12 months of age) reveal an even distribution of alkaline phosphatase along the enterocyte brush border, and a similar number and distribution of Paneth cells (Lysozyme) in the small intestine; and neuroendocrine (chromogranin A), goblet (PAS+), and proliferating (Ki67) cells in the colon. Scale bars, 50 µm. Representative microscopic views of the myenteric plexus of Ndrg4 +/+ and Ndrg4 −/− murine colonic sections labeled with either TuJ1 and S100 (B, n = 3) or HuC/D (C, n = 6) did not reveal structural or organizational differences in the ganglionic network of Ndrg4 +/+ and Ndrg4 −/− mice. Scale bars, 100 µm. Quantification of the enteric neuronal cell number (HuC/D) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4 +/+ and Ndrg4 −/− mice ( n = 6). Gastrointestinal motility assays reveal a similar whole‐gut transit time (E; n = 12 versus 12), with a similar weight per stool and stool water content of the fecal pellets (F, G; n = 11 versus 10), a comparable small intestinal transit (H; n = 10 versus 12) and colonic propulsion time (I; n = 7 versus 6) in Ndrg4 −/− compared to Ndrg4 +/+ mice. Data information: All data are presented as mean ± SEM, with P ‐values determined using a two‐tailed, unpaired t ‐test.

Techniques Used: Labeling, Mouse Assay, Two Tailed Test

33) Product Images from "High Glycolytic Activity Enhances Stem Cell Reprogramming of Fahd1-KO Mouse Embryonic Fibroblasts"

Article Title: High Glycolytic Activity Enhances Stem Cell Reprogramming of Fahd1-KO Mouse Embryonic Fibroblasts

Journal: Cells

doi: 10.3390/cells10082040

A high degree of mitochondrial biogenesis is maintained in Fahd1-KO iPSC-derived neurons. ( A ) Immunocytochemistry against the neuronal marker Tuj1 and the nuclei marker DAPI showed an efficient neuronal differentiation in both WT and Fahd1-KO iPSCs. ( B – D ) Relative mRNA expression of mt biogenesis markers, Pparg, Ppara, Slit1, Creb1, Nrf1, and Junk1. ( B ) Upregulation of mt biogenesis markers in iPSC-derived neurons relative to WT iPSCs. ( C ) Upregulation of mt biogenesis markers in Fahd1-KO iPSC-derived neurons relative to Fahd1-KO iPSCs. ( D ) Expression of mt biogenesis markers in neurons produced from Fahd1-KO and WT iPSCs. All the analyses were performed on iPSC-derived neurons after 20 days of differentiation, p
Figure Legend Snippet: A high degree of mitochondrial biogenesis is maintained in Fahd1-KO iPSC-derived neurons. ( A ) Immunocytochemistry against the neuronal marker Tuj1 and the nuclei marker DAPI showed an efficient neuronal differentiation in both WT and Fahd1-KO iPSCs. ( B – D ) Relative mRNA expression of mt biogenesis markers, Pparg, Ppara, Slit1, Creb1, Nrf1, and Junk1. ( B ) Upregulation of mt biogenesis markers in iPSC-derived neurons relative to WT iPSCs. ( C ) Upregulation of mt biogenesis markers in Fahd1-KO iPSC-derived neurons relative to Fahd1-KO iPSCs. ( D ) Expression of mt biogenesis markers in neurons produced from Fahd1-KO and WT iPSCs. All the analyses were performed on iPSC-derived neurons after 20 days of differentiation, p

Techniques Used: Derivative Assay, Immunocytochemistry, Marker, Expressing, Produced

Germ layer and neural differentiation showed similar cell fate between WT and Fahd1-KO iPSCs. ( A ) Phase contrast imaging showing typical iPSC-like round colonies when cultured on inactivated MEFs. ( B ) Immunofluorescence showing the expression of the pluripotency markers Oct4 and Sox2 in both WT and KO iPSCs. Cell nuclei were stained with DAPI. ( C ) After germ layer differentiation, immunofluorescence on the differentiated cells showed the expression of the endodermal marker AFP, the mesodermal marker SMA, and the ectodermal marker Tuj1. ( D ) Neural differentiation showed typical neural rosette formation both in WT and Fahd1-KO, as it is shown in the phase contrast images. ( E ) Relative mRNA expression by qRT-PCR of the pluripotency marker Nanog, and the neural progenitor markers Pax6 and Nestin in WT and Fahd1-KO MEFs, iPSCs, and neural progenitors. The results showed a downregulation of the pluripotency and an upregulation of neural markers similarly in both WT and Fahd1-KO. MEFs: mouse embryonic fibroblasts, iPSCs: induced pluripotent stem cells, WT: wild type, KO: knock-out, ND: not detected. Scale bars, 100 µm.
Figure Legend Snippet: Germ layer and neural differentiation showed similar cell fate between WT and Fahd1-KO iPSCs. ( A ) Phase contrast imaging showing typical iPSC-like round colonies when cultured on inactivated MEFs. ( B ) Immunofluorescence showing the expression of the pluripotency markers Oct4 and Sox2 in both WT and KO iPSCs. Cell nuclei were stained with DAPI. ( C ) After germ layer differentiation, immunofluorescence on the differentiated cells showed the expression of the endodermal marker AFP, the mesodermal marker SMA, and the ectodermal marker Tuj1. ( D ) Neural differentiation showed typical neural rosette formation both in WT and Fahd1-KO, as it is shown in the phase contrast images. ( E ) Relative mRNA expression by qRT-PCR of the pluripotency marker Nanog, and the neural progenitor markers Pax6 and Nestin in WT and Fahd1-KO MEFs, iPSCs, and neural progenitors. The results showed a downregulation of the pluripotency and an upregulation of neural markers similarly in both WT and Fahd1-KO. MEFs: mouse embryonic fibroblasts, iPSCs: induced pluripotent stem cells, WT: wild type, KO: knock-out, ND: not detected. Scale bars, 100 µm.

Techniques Used: Imaging, Cell Culture, Immunofluorescence, Expressing, Staining, Marker, Quantitative RT-PCR, Knock-Out

34) Product Images from "Exploration of Sensory and Spinal Neurons Expressing GRP in Itch and Pain"

Article Title: Exploration of Sensory and Spinal Neurons Expressing GRP in Itch and Pain

Journal: bioRxiv

doi: 10.1101/472886

Opto-activation of Grp + sensory neuron skin fibers evokes itch behavior. ( A ) Schematic of Grp Cre KI mating with Ai32 ChR2-eYFP line to produce Grp ChR2 mice. ( B ) IHC Image of eYFP expression in Grp ChR2 DRG. Scale bar in B , 50 μm. ( C ) IHC images of eYFP and ßlll-Tubulin in Grp ChR2 nape skin. Dashed lines indicate epidermal/dermal boundary. Scale bar in C , 100 μm. ( D ) Optical parameters of skin fiber stimulation Grp ChR2 and Grp WT mice. ( E ) Raster plot of scratching behavior induced by light stimulation of skin in Grp ChR2 and Grp WT mice. ( F ) Snapshots of Grp ChR2 and Grp WT mice with light off or on. Arrow indicates hind paw scratching the nape when light is on. ( G ) Total number of scratches during 5-min light stimulation experiment in Grp WT , Grp ChR2 , Grp ChR2 morphine-treated and Grp ChR2 BB-sap-treated mice. ( H ) n = 8 – 10 mice, one-way ANOVA with Tukey post hoc , *** p
Figure Legend Snippet: Opto-activation of Grp + sensory neuron skin fibers evokes itch behavior. ( A ) Schematic of Grp Cre KI mating with Ai32 ChR2-eYFP line to produce Grp ChR2 mice. ( B ) IHC Image of eYFP expression in Grp ChR2 DRG. Scale bar in B , 50 μm. ( C ) IHC images of eYFP and ßlll-Tubulin in Grp ChR2 nape skin. Dashed lines indicate epidermal/dermal boundary. Scale bar in C , 100 μm. ( D ) Optical parameters of skin fiber stimulation Grp ChR2 and Grp WT mice. ( E ) Raster plot of scratching behavior induced by light stimulation of skin in Grp ChR2 and Grp WT mice. ( F ) Snapshots of Grp ChR2 and Grp WT mice with light off or on. Arrow indicates hind paw scratching the nape when light is on. ( G ) Total number of scratches during 5-min light stimulation experiment in Grp WT , Grp ChR2 , Grp ChR2 morphine-treated and Grp ChR2 BB-sap-treated mice. ( H ) n = 8 – 10 mice, one-way ANOVA with Tukey post hoc , *** p

Techniques Used: Activation Assay, Mouse Assay, Immunohistochemistry, Expressing

( A ) IHC images of eYFP, CGRP and IB4 in Grp ChR2 DRG. Scale bar in A , 50 μm. ( B ) eYFP image from Grp WT ; Ai32 DRG. Scale bar in B , 50 μm. ( C ) IHC image of eYFP, βlII-Tubulin and DAPI in Grp WT ; Ai32 nape skin. Scale bar in C , 100 μm. ( D ) IHC image of eYFP and ßIII-Tubulin in Grp ChR2 cheek skin. Scale bar in D , 100 μm. ( E ) IHC images of eYFP, CGRP and DAPI merge in Grp ChR2 glabrous skin. Scale bar in E , 100 μm. ( F ) IHC image of eYFP and DiI in Grp ChR2 DRG or TG 10 days after i.d. nape or cheek injection of DiI tracer. Scale bar in F , 50 μm.
Figure Legend Snippet: ( A ) IHC images of eYFP, CGRP and IB4 in Grp ChR2 DRG. Scale bar in A , 50 μm. ( B ) eYFP image from Grp WT ; Ai32 DRG. Scale bar in B , 50 μm. ( C ) IHC image of eYFP, βlII-Tubulin and DAPI in Grp WT ; Ai32 nape skin. Scale bar in C , 100 μm. ( D ) IHC image of eYFP and ßIII-Tubulin in Grp ChR2 cheek skin. Scale bar in D , 100 μm. ( E ) IHC images of eYFP, CGRP and DAPI merge in Grp ChR2 glabrous skin. Scale bar in E , 100 μm. ( F ) IHC image of eYFP and DiI in Grp ChR2 DRG or TG 10 days after i.d. nape or cheek injection of DiI tracer. Scale bar in F , 50 μm.

Techniques Used: Immunohistochemistry, Injection

35) Product Images from "Interleukin-6 promotes microtubule stability in axons via Stat3 protein–protein interactions"

Article Title: Interleukin-6 promotes microtubule stability in axons via Stat3 protein–protein interactions

Journal: iScience

doi: 10.1016/j.isci.2021.103141

IL-6 deficiency induces tubulin disorganization in RGC axons (A and B) Representative confocal micrographs (100X) of α-tubulin (A; red) and β-tubulin (βTubIII; B; green) immunolabeling in peripheral (left) and central (middle) regions of wholemount retina from WT (top) and IL-6−/− (bottom) mice. Arrowheads indicate regions with aggregated labeling. Boxed regions were zoomed to highlight aggregations (right). Scale = 20 μm. (C) Immunoblotting (top) and densitometric quantification (bottom) of α-tubulin (50 kDa) and β-tubulin (55 kDa) normalized to GAPDH (36 kDa) in retina and optic nerve from WT and IL-6−/− mice (n = 3). Error bars = standard deviation.
Figure Legend Snippet: IL-6 deficiency induces tubulin disorganization in RGC axons (A and B) Representative confocal micrographs (100X) of α-tubulin (A; red) and β-tubulin (βTubIII; B; green) immunolabeling in peripheral (left) and central (middle) regions of wholemount retina from WT (top) and IL-6−/− (bottom) mice. Arrowheads indicate regions with aggregated labeling. Boxed regions were zoomed to highlight aggregations (right). Scale = 20 μm. (C) Immunoblotting (top) and densitometric quantification (bottom) of α-tubulin (50 kDa) and β-tubulin (55 kDa) normalized to GAPDH (36 kDa) in retina and optic nerve from WT and IL-6−/− mice (n = 3). Error bars = standard deviation.

Techniques Used: Immunolabeling, Mouse Assay, Labeling, Standard Deviation

IL-6 stabilizes microtubules and preserves neurite structure (A) Representative confocal images (40X) of β-III-Tubulin immunolabeling (green) and DAPI counterstain (blue) in primary, purified RGCs treated for 1 h with vehicle or 1mM colchicine following 1 h pre-treatment with vehicle, 1ng/mL rIL-6 or 1ng/mL rIL-6 + 10ng/mL nAb. Scale = 50 μm. (B) Corresponding Scholl analysis tracings to RGCs pictured in (A). (C) Scholl function depicting the mean number of intersections between β-tubulin+ neurites and Scholl radius intervals originating from the center of RGC soma for each condition. (D) Scholl intersection profile (SIP) quantified as the mean area under the curve (AUC) for each condition. Asterisk = p
Figure Legend Snippet: IL-6 stabilizes microtubules and preserves neurite structure (A) Representative confocal images (40X) of β-III-Tubulin immunolabeling (green) and DAPI counterstain (blue) in primary, purified RGCs treated for 1 h with vehicle or 1mM colchicine following 1 h pre-treatment with vehicle, 1ng/mL rIL-6 or 1ng/mL rIL-6 + 10ng/mL nAb. Scale = 50 μm. (B) Corresponding Scholl analysis tracings to RGCs pictured in (A). (C) Scholl function depicting the mean number of intersections between β-tubulin+ neurites and Scholl radius intervals originating from the center of RGC soma for each condition. (D) Scholl intersection profile (SIP) quantified as the mean area under the curve (AUC) for each condition. Asterisk = p

Techniques Used: Immunolabeling, Purification

36) Product Images from "Axons in the Chick Embryo Follow Soft Pathways Through Developing Somite Segments"

Article Title: Axons in the Chick Embryo Follow Soft Pathways Through Developing Somite Segments

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2022.917589

Motor axons grow out of the neural tube in a segmented fashion. (A) Schematic representation of the developing peripheral nervous system in chick embryo embryonic day 3 (E3), stage HH18. Motor neurons project their axon out of the neural tube, in the sclerotome, along a ventral-lateral trajectory. The frontal plane (in blue) is used to study segmentation of outgrowing motor axons. (D) dorsal; V: ventral. (B) Immunohistochemistry on a frontal section of a chick embryo showing motor axons (Tubulin III β, red) growing out of the neural tube (nt) and cell nuclei (Hoechst, blue) in somite segments. Scale bar = 100 μm dm: dermomyotome; sc: sclerotome; nc: notochord.
Figure Legend Snippet: Motor axons grow out of the neural tube in a segmented fashion. (A) Schematic representation of the developing peripheral nervous system in chick embryo embryonic day 3 (E3), stage HH18. Motor neurons project their axon out of the neural tube, in the sclerotome, along a ventral-lateral trajectory. The frontal plane (in blue) is used to study segmentation of outgrowing motor axons. (D) dorsal; V: ventral. (B) Immunohistochemistry on a frontal section of a chick embryo showing motor axons (Tubulin III β, red) growing out of the neural tube (nt) and cell nuclei (Hoechst, blue) in somite segments. Scale bar = 100 μm dm: dermomyotome; sc: sclerotome; nc: notochord.

Techniques Used: Immunohistochemistry

37) Product Images from "Microdomains form on the luminal face of neuronal extracellular vesicle membranes"

Article Title: Microdomains form on the luminal face of neuronal extracellular vesicle membranes

Journal: Scientific Reports

doi: 10.1038/s41598-020-68436-x

( A ) Cortical neurons were fixed and immunostained for neuron-specific β3-tubulin (Tuj). ( B ) Transmission electron micrograph of EVs purified from neuron-conditioned media observed by negative staining. ( C ) Representative image of EVs imaged by cryoEM. ( D ) Schematic of procedures used to concentrate EVs by differential centrifugation (dUC). ( E ) Frequency distribution of dUC EV diameter determined by cryoEM. Dotted line separates small and medium-sized EVs. ( F ) Western blotting of centrifugation fractions prepared as in D. CL, cell lysate. Original unprocessed blots can be found in Supplementary Fig. S1 . ( G ) Schematic of procedures used to concentrate EVs by ultrafiltration (UF). ( H ) Frequency distribution of UF EV diameter determined by cryoEM. Dotted line separates small and medium-sized EVs. ( I ) Western blotting of ultrafiltration fractions prepared as in G. CL, cell lysate. Original unprocessed blots can be found in Supplementary Fig. S1 .
Figure Legend Snippet: ( A ) Cortical neurons were fixed and immunostained for neuron-specific β3-tubulin (Tuj). ( B ) Transmission electron micrograph of EVs purified from neuron-conditioned media observed by negative staining. ( C ) Representative image of EVs imaged by cryoEM. ( D ) Schematic of procedures used to concentrate EVs by differential centrifugation (dUC). ( E ) Frequency distribution of dUC EV diameter determined by cryoEM. Dotted line separates small and medium-sized EVs. ( F ) Western blotting of centrifugation fractions prepared as in D. CL, cell lysate. Original unprocessed blots can be found in Supplementary Fig. S1 . ( G ) Schematic of procedures used to concentrate EVs by ultrafiltration (UF). ( H ) Frequency distribution of UF EV diameter determined by cryoEM. Dotted line separates small and medium-sized EVs. ( I ) Western blotting of ultrafiltration fractions prepared as in G. CL, cell lysate. Original unprocessed blots can be found in Supplementary Fig. S1 .

Techniques Used: Transmission Assay, Purification, Negative Staining, Centrifugation, Western Blot

38) Product Images from "The PARK10 gene USP24 is a negative regulator of autophagy and ULK1 protein stability"

Article Title: The PARK10 gene USP24 is a negative regulator of autophagy and ULK1 protein stability

Journal: Autophagy

doi: 10.1080/15548627.2019.1598754

USP24 regulates autophagy and neurite length in human iPSC-derived dopaminergic neurons. (a) Western blot illustrating the changes in LC3-II, NBR1, and phospho-ATG14 (P-ATG14) in human iPSC-derived dopaminergic neurons following USP24 knockdown. Dopaminergic precursor cells were differentiated into dopaminergic neurons for 2 wk, transduced with indicated lentiviral shRNAs and evaluated after an additional week in culture. (b) Quantification of USP24:tubulin levels from figure (a). (c) Quantification of LC3-II:tubulin levels from figure (a) (d) Quantification of NBR1:tubulin levels from figure (a) (2 independent experiments; 4–6 replicates) (e and f) Levels of LC3-II following USP24 knockdown are further increased with 100 μM chloroquine (chq) treatment (overnight). (e) Western blot illustrating change in LC3-II levels in human iPSC-derived dopaminergic neurons following USP24 knockdown. (f) Quantification of LC3-II:loading control from figure (e) (4–6 replicates). (g) Representative images of human iPSC-derived neurons at 6 wk after lentiviral transduction, showing increased neurite density in cultures with USP24 knockdown. Cells were transduced with indicated shRNAs; after additional 6 wk cultures were fixed and stained with antibodies against TUBB3/tubulin B3/TUJ1; all neurons) and TH (tyrosine hydroxylase; dopaminergic neurons). Images were acquired at 20X; bar: 25 μm. (h) Quantification of length of neuronal processes over time after lentiviral transduction with indicated shRNAs. All data are normalized to nt shRNA at 1-week time point. Statistical analysis at 1-week time point is presented in Figure S4 and at 6-weeks in (i-j). (i) Quantification of neurite length:neuron from figure (h) at 6-week time point. Data are normalized to nt shRNA at 6-week time point. (j) Quantification of neurite length:neuron for the dopaminergic neurons (TH positive) at 6-week time point. Data are normalized to nt shRNA at 6-week time point. All data are presented as ±SEM. *p
Figure Legend Snippet: USP24 regulates autophagy and neurite length in human iPSC-derived dopaminergic neurons. (a) Western blot illustrating the changes in LC3-II, NBR1, and phospho-ATG14 (P-ATG14) in human iPSC-derived dopaminergic neurons following USP24 knockdown. Dopaminergic precursor cells were differentiated into dopaminergic neurons for 2 wk, transduced with indicated lentiviral shRNAs and evaluated after an additional week in culture. (b) Quantification of USP24:tubulin levels from figure (a). (c) Quantification of LC3-II:tubulin levels from figure (a) (d) Quantification of NBR1:tubulin levels from figure (a) (2 independent experiments; 4–6 replicates) (e and f) Levels of LC3-II following USP24 knockdown are further increased with 100 μM chloroquine (chq) treatment (overnight). (e) Western blot illustrating change in LC3-II levels in human iPSC-derived dopaminergic neurons following USP24 knockdown. (f) Quantification of LC3-II:loading control from figure (e) (4–6 replicates). (g) Representative images of human iPSC-derived neurons at 6 wk after lentiviral transduction, showing increased neurite density in cultures with USP24 knockdown. Cells were transduced with indicated shRNAs; after additional 6 wk cultures were fixed and stained with antibodies against TUBB3/tubulin B3/TUJ1; all neurons) and TH (tyrosine hydroxylase; dopaminergic neurons). Images were acquired at 20X; bar: 25 μm. (h) Quantification of length of neuronal processes over time after lentiviral transduction with indicated shRNAs. All data are normalized to nt shRNA at 1-week time point. Statistical analysis at 1-week time point is presented in Figure S4 and at 6-weeks in (i-j). (i) Quantification of neurite length:neuron from figure (h) at 6-week time point. Data are normalized to nt shRNA at 6-week time point. (j) Quantification of neurite length:neuron for the dopaminergic neurons (TH positive) at 6-week time point. Data are normalized to nt shRNA at 6-week time point. All data are presented as ±SEM. *p

Techniques Used: Derivative Assay, Western Blot, Transduction, Staining, shRNA

39) Product Images from "Progranulin-mediated deficiency of cathepsin D results in FTD and NCL-like phenotypes in neurons derived from FTD patients"

Article Title: Progranulin-mediated deficiency of cathepsin D results in FTD and NCL-like phenotypes in neurons derived from FTD patients

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddx364

iPSC-derived PGRN mutant neurons have reduced cathepsin D activity. ( A ) Cathepsin D activity in PGRN WT and mutant cortical neuron lysates (days 35 and 100 post-differentiation) ( n = 3). Immature and mature ( B ) cathepsin D and ( C ) cathepsin B expression in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) shown by Western blot analysis using β-III tubulin, a neuronal marker, as a loading control. Quantification of ( D ) mature and ( E ) immature cathepsin D expression in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) ( n = 3). ( F ) Ratio of mature: immature cathepsin D in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) ( n = 3). ( G ) Cathepsin D activity per mature cathepsin D expression in PGRN WT and mutant neuron lysates (35 and 100 days post-differentiation) ( n = 3). ( H ) Co-immunoprecipitation assay in HEK293 cells co-expressing cathepsin D-V5 and PGRN show cathepsin D and PGRN interaction ( n = 3). Student’s t -test, unpaired, two-tailed statistical analysis was performed. Error bars represent S.E.M. of total experiments, n.s., not significant, * P
Figure Legend Snippet: iPSC-derived PGRN mutant neurons have reduced cathepsin D activity. ( A ) Cathepsin D activity in PGRN WT and mutant cortical neuron lysates (days 35 and 100 post-differentiation) ( n = 3). Immature and mature ( B ) cathepsin D and ( C ) cathepsin B expression in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) shown by Western blot analysis using β-III tubulin, a neuronal marker, as a loading control. Quantification of ( D ) mature and ( E ) immature cathepsin D expression in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) ( n = 3). ( F ) Ratio of mature: immature cathepsin D in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) ( n = 3). ( G ) Cathepsin D activity per mature cathepsin D expression in PGRN WT and mutant neuron lysates (35 and 100 days post-differentiation) ( n = 3). ( H ) Co-immunoprecipitation assay in HEK293 cells co-expressing cathepsin D-V5 and PGRN show cathepsin D and PGRN interaction ( n = 3). Student’s t -test, unpaired, two-tailed statistical analysis was performed. Error bars represent S.E.M. of total experiments, n.s., not significant, * P

Techniques Used: Derivative Assay, Mutagenesis, Activity Assay, Expressing, Western Blot, Marker, Co-Immunoprecipitation Assay, Two Tailed Test

iPSC-derived PGRN mutant neurons have impaired lysosomal function. ( A ) Lysosomal proteolysis measured in PGRN WT and mutant cortical neurons (day 35 post-differentiation) through pulse-chase analysis ( n = 6). ( B ) LAMP1 and LAMP2 expression in PGRN WT and mutant neuron lysates (day 35 post-differentiation) shown by Western blot analysis using β-III tubulin, a neuronal marker, as a loading control. Quantification of ( C ) LAMP1 and ( D ) LAMP2 expression in PGRN WT and mutant neuron lysates (day 35 post-differentiation) ( n = 3). ( E ) Immunofluorescence of LAMP1 in PGRN WT and mutant neurons (day 35 post-differentiation). ( F ) Quantification of LAMP1 immunofluorescence intensity in PGRN WT and mutant neurons (day 35 post-differentiation) ( n = 3, 95–114 cells/experiment). Student’s t -test, unpaired, two-tailed statistical analysis was performed. Error bars represent S.E.M. of total experiments, n.s., not significant, * P
Figure Legend Snippet: iPSC-derived PGRN mutant neurons have impaired lysosomal function. ( A ) Lysosomal proteolysis measured in PGRN WT and mutant cortical neurons (day 35 post-differentiation) through pulse-chase analysis ( n = 6). ( B ) LAMP1 and LAMP2 expression in PGRN WT and mutant neuron lysates (day 35 post-differentiation) shown by Western blot analysis using β-III tubulin, a neuronal marker, as a loading control. Quantification of ( C ) LAMP1 and ( D ) LAMP2 expression in PGRN WT and mutant neuron lysates (day 35 post-differentiation) ( n = 3). ( E ) Immunofluorescence of LAMP1 in PGRN WT and mutant neurons (day 35 post-differentiation). ( F ) Quantification of LAMP1 immunofluorescence intensity in PGRN WT and mutant neurons (day 35 post-differentiation) ( n = 3, 95–114 cells/experiment). Student’s t -test, unpaired, two-tailed statistical analysis was performed. Error bars represent S.E.M. of total experiments, n.s., not significant, * P

Techniques Used: Derivative Assay, Mutagenesis, Pulse Chase, Expressing, Western Blot, Marker, Immunofluorescence, Two Tailed Test

FTD-linked PGRN mutant neurons exhibit both FTD and NCL-like pathology. ( A ) PGRN expression in PGRN WT and mutant iPSC-derived neurons (day 35 post-differentiation) using NSE, a neuronal marker, as a loading control. ( B) Immunofluorescence of TDP-43 and neuronal marker β-III tubulin in PGRN WT and mutant neuronal cultures (day 35 post-differentiation). PGRN mutant neurons have decreased nuclear TDP-43 (white arrows) as compared to PGRN WT neurons (yellow arrows). ( C ) Insoluble TDP-43 in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) shown by Western blot analysis using vinculin as a loading control. ( D ) Quantification of insoluble TDP-43 in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) ( n = 3). ( E ) Electron dense vesicles in PGRN WT and mutant neurons (day 130 post-differentiation) visualized through electron microscopy analysis. Scale bar, 500 nM. ( F ) Quantification of electron-dense vesicle size in PGRN WT and mutant neurons (day 130 post-differentiation) ( n = 6–8 cells/line). ( G ) Autofluorescent puncta in PGRN WT and mutant neurons (day 130 post-differentiation). ( H ) Quantification of lipofuscin puncta/cell in PGRN WT and mutant neurons (day 130 post-differentiation) ( n = 3, 30–50 cells/experiment). Electron microscopy analysis of PGRN mutant neurons (day 130 post-differentiation) showed ( I ) fingerprint-like profiles and ( J ) granular osmiophilic deposits ( n = 6–8 cells/line). Scale bar, 500 nM. Student’s t -test, unpaired, two-tailed statistical analysis was performed. Error bars represent S.E.M. of total experiments. n.s., not significant, * P
Figure Legend Snippet: FTD-linked PGRN mutant neurons exhibit both FTD and NCL-like pathology. ( A ) PGRN expression in PGRN WT and mutant iPSC-derived neurons (day 35 post-differentiation) using NSE, a neuronal marker, as a loading control. ( B) Immunofluorescence of TDP-43 and neuronal marker β-III tubulin in PGRN WT and mutant neuronal cultures (day 35 post-differentiation). PGRN mutant neurons have decreased nuclear TDP-43 (white arrows) as compared to PGRN WT neurons (yellow arrows). ( C ) Insoluble TDP-43 in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) shown by Western blot analysis using vinculin as a loading control. ( D ) Quantification of insoluble TDP-43 in PGRN WT and mutant neuron lysates (days 35 and 100 post-differentiation) ( n = 3). ( E ) Electron dense vesicles in PGRN WT and mutant neurons (day 130 post-differentiation) visualized through electron microscopy analysis. Scale bar, 500 nM. ( F ) Quantification of electron-dense vesicle size in PGRN WT and mutant neurons (day 130 post-differentiation) ( n = 6–8 cells/line). ( G ) Autofluorescent puncta in PGRN WT and mutant neurons (day 130 post-differentiation). ( H ) Quantification of lipofuscin puncta/cell in PGRN WT and mutant neurons (day 130 post-differentiation) ( n = 3, 30–50 cells/experiment). Electron microscopy analysis of PGRN mutant neurons (day 130 post-differentiation) showed ( I ) fingerprint-like profiles and ( J ) granular osmiophilic deposits ( n = 6–8 cells/line). Scale bar, 500 nM. Student’s t -test, unpaired, two-tailed statistical analysis was performed. Error bars represent S.E.M. of total experiments. n.s., not significant, * P

Techniques Used: Mutagenesis, Expressing, Derivative Assay, Marker, Immunofluorescence, Western Blot, Electron Microscopy, Two Tailed Test

40) Product Images from "Quantitative proteomics identifies proteins that resist translational repression and become dysregulated in ALS-FUS"

Article Title: Quantitative proteomics identifies proteins that resist translational repression and become dysregulated in ALS-FUS

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddz048

Newly synthesized COPBI protein is reduced in mutant FUS expressing cells under stress. (A, B) Western blot analysis of immunoprecipitated COPBI from arsenite stressed SK-N-AS FUS WT and R495X cells. (A) Input lanes show similar levels of COPBI protein in cell lysates, whereas AHA-labeled COPBI protein is below the limit of detection in the whole cell lysate (the shadow of a band at the top of the image corresponds to a protein of higher molecular weight than COPBI) (left). Anti-COPBI antibody (top) confirms that similar levels of COPBI were isolated from the FUS WT and R495X lysates (right). Immunoblotting the same blot with a streptavidin secondary antibody against biotin-conjugated AHA-labeled proteins (bottom) shows this band contains less newly synthesized COPBI protein in FUS R495X cells under arsenite (SA) stress. (B) Quantification of (A), n = 3 independent biological experiments (Student’s t -test, ** P = 0.002). (C, D) Western analysis of neurons derived from transgenic FUS R495X (RX) mice and NTG littermates. (C) Representative Western blot with anti-COPBI and anti-TUBB3 (Tuj1; loading control) antibodies. (D) Quantification of (C), 3 biological experiments. (E–G) Western analysis of other COPI subunits. (E) Representative Western blot of NTG and FUS R495X neurons with anti-COPA, COPGI and TUJ1 antibodies. (F, G) Quantification of (E), 3 or 4 biological experiments for (F) and (G), respectively. (H) Quantitative PCR analysis of COPBI mRNA levels in NTG and FUS R495X neurons (4 biological experiments). (D, F–H) Data were analyzed by two-way ANOVA with Tukey post hoc testing for multiple comparisons, and error bars reflect standard error of the mean. Statistically significant comparisons are represented by * P
Figure Legend Snippet: Newly synthesized COPBI protein is reduced in mutant FUS expressing cells under stress. (A, B) Western blot analysis of immunoprecipitated COPBI from arsenite stressed SK-N-AS FUS WT and R495X cells. (A) Input lanes show similar levels of COPBI protein in cell lysates, whereas AHA-labeled COPBI protein is below the limit of detection in the whole cell lysate (the shadow of a band at the top of the image corresponds to a protein of higher molecular weight than COPBI) (left). Anti-COPBI antibody (top) confirms that similar levels of COPBI were isolated from the FUS WT and R495X lysates (right). Immunoblotting the same blot with a streptavidin secondary antibody against biotin-conjugated AHA-labeled proteins (bottom) shows this band contains less newly synthesized COPBI protein in FUS R495X cells under arsenite (SA) stress. (B) Quantification of (A), n = 3 independent biological experiments (Student’s t -test, ** P = 0.002). (C, D) Western analysis of neurons derived from transgenic FUS R495X (RX) mice and NTG littermates. (C) Representative Western blot with anti-COPBI and anti-TUBB3 (Tuj1; loading control) antibodies. (D) Quantification of (C), 3 biological experiments. (E–G) Western analysis of other COPI subunits. (E) Representative Western blot of NTG and FUS R495X neurons with anti-COPA, COPGI and TUJ1 antibodies. (F, G) Quantification of (E), 3 or 4 biological experiments for (F) and (G), respectively. (H) Quantitative PCR analysis of COPBI mRNA levels in NTG and FUS R495X neurons (4 biological experiments). (D, F–H) Data were analyzed by two-way ANOVA with Tukey post hoc testing for multiple comparisons, and error bars reflect standard error of the mean. Statistically significant comparisons are represented by * P

Techniques Used: Synthesized, Mutagenesis, Expressing, Western Blot, Immunoprecipitation, Labeling, Molecular Weight, Isolation, Derivative Assay, Transgenic Assay, Mouse Assay, Real-time Polymerase Chain Reaction

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    BioLegend mouse anti tubulin β3
    related to Figure 3. Additional Evidence that ZDHHC17 is a Major DLK PAT. A: Quantified intensities of ZDHHC17 protein levels from Fig 3A , normalized to <t>tubulin,</t> confirm efficacy of Zdhhc17 shRNAs. N=9-10 individual cultures per condition; ***:p
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    related to Figure 3. Additional Evidence that ZDHHC17 is a Major DLK PAT. A: Quantified intensities of ZDHHC17 protein levels from Fig 3A , normalized to tubulin, confirm efficacy of Zdhhc17 shRNAs. N=9-10 individual cultures per condition; ***:p

    Journal: bioRxiv

    Article Title: Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17

    doi: 10.1101/2020.09.01.276287

    Figure Lengend Snippet: related to Figure 3. Additional Evidence that ZDHHC17 is a Major DLK PAT. A: Quantified intensities of ZDHHC17 protein levels from Fig 3A , normalized to tubulin, confirm efficacy of Zdhhc17 shRNAs. N=9-10 individual cultures per condition; ***:p

    Article Snippet: AntibodiesPrimary antibodies used were as follows: Rabbit anti-phospho c-Jun S63 II (Cell Signaling Technology, #9261, 1:100, used for sensory neuron experiments), Rabbit anti-phospho c-Jun Ser-73 (Cell Signaling Technology, #3270, used for RGC experiments); rabbit anti-DLK/MAP3K12 (Sigma/ Prestige, #HPA039936, used for western blotting); rabbit anti-DLK/MAP3K12 (Thermofisher Scientific, #PA5-32173, used for immunostaining); mouse anti-GFP (Life Technologies, #A11120, clone 3E6 IgG2a, 1:250), rabbit anti-GFP (Life Technologies, #A11122), mouse anti-Tubulin β3 (BioLegend, TUJ1, Ig2a, Covance catalog# MMS-435P, 1:1000), mouse anti-Myc 9E10 (University of Pennsylvania Cell Center, Catalog #3207), mouse anti-HA11 (BioLegend, 901502, IgG1, 1:500), rabbit anti-HA (Cell Signaling Technology, #3724), rabbit anti-myc (Cell Signaling Technology, #2278), mouse anti-tubulin (Millipore Sigma, Catalog #T7451), sheep anti-NGF (CedarLane, catalog #CLMCNET-031).

    Techniques:

    ZDHHC17 Localizes to the Somatic Golgi in Mammalian Sensory Neurons. A: Images of cultured DRG neurons coinfected with lentiviruses to express GFP and HA-ZDHHC17, immunostained with the indicated antibodies. HA-ZDHHC17 is detected in the neuronal soma but not axons. Images are representative of 10 individual neurons examined. Scale bar: 20 μm. B: Western blots of somata plus axons (Soma + Axon) or distal axons only (‘Axon’) fractions from 3 sets of DRG neurons cultured in microfluidic chambers, blotted with the indicated antibodies. The ‘Axon’ chamber was lysed in 1/12 of the volume used for the ‘Soma + Axon’ chamber to account for the lower amount of material in the former compartment. C: Quantified data from n=4 determinations per condition of ZDHHC17 protein level, normalized to tubulin, for each of the indicated subcellular chambers from B . **: p

    Journal: bioRxiv

    Article Title: Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17

    doi: 10.1101/2020.09.01.276287

    Figure Lengend Snippet: ZDHHC17 Localizes to the Somatic Golgi in Mammalian Sensory Neurons. A: Images of cultured DRG neurons coinfected with lentiviruses to express GFP and HA-ZDHHC17, immunostained with the indicated antibodies. HA-ZDHHC17 is detected in the neuronal soma but not axons. Images are representative of 10 individual neurons examined. Scale bar: 20 μm. B: Western blots of somata plus axons (Soma + Axon) or distal axons only (‘Axon’) fractions from 3 sets of DRG neurons cultured in microfluidic chambers, blotted with the indicated antibodies. The ‘Axon’ chamber was lysed in 1/12 of the volume used for the ‘Soma + Axon’ chamber to account for the lower amount of material in the former compartment. C: Quantified data from n=4 determinations per condition of ZDHHC17 protein level, normalized to tubulin, for each of the indicated subcellular chambers from B . **: p

    Article Snippet: AntibodiesPrimary antibodies used were as follows: Rabbit anti-phospho c-Jun S63 II (Cell Signaling Technology, #9261, 1:100, used for sensory neuron experiments), Rabbit anti-phospho c-Jun Ser-73 (Cell Signaling Technology, #3270, used for RGC experiments); rabbit anti-DLK/MAP3K12 (Sigma/ Prestige, #HPA039936, used for western blotting); rabbit anti-DLK/MAP3K12 (Thermofisher Scientific, #PA5-32173, used for immunostaining); mouse anti-GFP (Life Technologies, #A11120, clone 3E6 IgG2a, 1:250), rabbit anti-GFP (Life Technologies, #A11122), mouse anti-Tubulin β3 (BioLegend, TUJ1, Ig2a, Covance catalog# MMS-435P, 1:1000), mouse anti-Myc 9E10 (University of Pennsylvania Cell Center, Catalog #3207), mouse anti-HA11 (BioLegend, 901502, IgG1, 1:500), rabbit anti-HA (Cell Signaling Technology, #3724), rabbit anti-myc (Cell Signaling Technology, #2278), mouse anti-tubulin (Millipore Sigma, Catalog #T7451), sheep anti-NGF (CedarLane, catalog #CLMCNET-031).

    Techniques: Cell Culture, Western Blot

    R101Q mutation inhibits NLGN4-induced excitatory synapse formation in NSC-derived human neurons. A , NSCs were induced from H1-ES cells by dual-SMAD inhibition. The NSC-derived neurons were cocultured with glia, infected with lentivirus-expressing transgenes, and analyzed at indicated time points (arrowheads). B , Design of lentiviral vectors expressing NLGN4 WT or R101Q variant under hSyn1 promoter followed by an IRES-EGFP construct. An EGFP-only vector was used as infection control (Ctrl). C , Images illustrate cellular identities, when immunolabeled for ES cell marker (Oct3/4), NSC marker (Nestin), or neuronal marker (Dcx), at different stages of the differentiation protocol. DAPI was used for nuclear stain. At day 60, elaborate neuronal morphology was confirmed by EGFP signal. Arrowheads point at neurons (yellow) and glial cells (white) in the culture. D , Sample images (top) depict NSC-derived neurons expressing NLGN4 WT versus R101Q variant, when immunostained for cytoskeletal marker Tuj1 and synaptic marker Synapsin. Bar graphs (below) represent normalized counts of cell bodies per field of view (left), total neurite volume (middle), and integrated signal intensity of Synapsin (right), for WT versus R101Q conditions. E , Representative images (top) of MAP2-positive dendritic segments counterstained for Synapsin, and summary graphs (bottom) of the density and size of Synapsin puncta, as quantified from cells expressing NLGN4 WT versus R101Q mutant. F , Example images (left) of MAP2-positive dendritic branches from cells in control, NLGN4 WT, and R101Q mutant conditions, as counterstained for an inhibitory synapse marker vGAT. Summary graphs (right) of the density and size of vGAT puncta. G , Same as F , except the neurons were immunolabeled for VGluT, an excitatory synapse marker. Averages indicate the mean ± SEM, with the total number of fields of view analyzed/number of batches. Statistical significance was tested by two-tailed, unpaired, Student's t test. *** p

    Journal: The Journal of Neuroscience

    Article Title: An Autism-Associated Mutation Impairs Neuroligin-4 Glycosylation and Enhances Excitatory Synaptic Transmission in Human Neurons

    doi: 10.1523/JNEUROSCI.0404-20.2020

    Figure Lengend Snippet: R101Q mutation inhibits NLGN4-induced excitatory synapse formation in NSC-derived human neurons. A , NSCs were induced from H1-ES cells by dual-SMAD inhibition. The NSC-derived neurons were cocultured with glia, infected with lentivirus-expressing transgenes, and analyzed at indicated time points (arrowheads). B , Design of lentiviral vectors expressing NLGN4 WT or R101Q variant under hSyn1 promoter followed by an IRES-EGFP construct. An EGFP-only vector was used as infection control (Ctrl). C , Images illustrate cellular identities, when immunolabeled for ES cell marker (Oct3/4), NSC marker (Nestin), or neuronal marker (Dcx), at different stages of the differentiation protocol. DAPI was used for nuclear stain. At day 60, elaborate neuronal morphology was confirmed by EGFP signal. Arrowheads point at neurons (yellow) and glial cells (white) in the culture. D , Sample images (top) depict NSC-derived neurons expressing NLGN4 WT versus R101Q variant, when immunostained for cytoskeletal marker Tuj1 and synaptic marker Synapsin. Bar graphs (below) represent normalized counts of cell bodies per field of view (left), total neurite volume (middle), and integrated signal intensity of Synapsin (right), for WT versus R101Q conditions. E , Representative images (top) of MAP2-positive dendritic segments counterstained for Synapsin, and summary graphs (bottom) of the density and size of Synapsin puncta, as quantified from cells expressing NLGN4 WT versus R101Q mutant. F , Example images (left) of MAP2-positive dendritic branches from cells in control, NLGN4 WT, and R101Q mutant conditions, as counterstained for an inhibitory synapse marker vGAT. Summary graphs (right) of the density and size of vGAT puncta. G , Same as F , except the neurons were immunolabeled for VGluT, an excitatory synapse marker. Averages indicate the mean ± SEM, with the total number of fields of view analyzed/number of batches. Statistical significance was tested by two-tailed, unpaired, Student's t test. *** p

    Article Snippet: Primary antibodies for immunolabeling included mouse anti-HA (1:1000; catalog #h3663, Sigma-Aldrich), rabbit anti-calnexin (1:500; catalog #PA5-34 754, Thermo Fisher Scientific), rabbit anti-GM130 (1:300; catalog #A5344, ABClonal), chicken anti-GFP (1:500; catalog #GFP-1020, Aves Labs), mouse anti-Oct3/4 (1:200; catalog #sc-5279, Santa Cruz Biotechnology), mouse anti-Nestin (1:500; catalog #MAB1259, R & D Systems), goat anti-Dcx (1:500; mixture of catalog #sc-8066 and #8067, Santa Cruz Biotechnology), mouse anti-Tuj1 (1:1000; catalog # 801202, BioLegend), chicken anti-MAP2 (1:400; catalog #ab5392, Abcam), mouse anti-MAP2 (1:500; catalog #M1406, Sigma-Aldrich), rabbit anti-Synapsin1/2 (1:500; catalog #106002, Synaptic Systems), rabbit anti-vGAT (1:200; catalog #131003, Synaptic Systems), rabbit anti-VGluT1/2 (1:500; YENZYM ANTIBODIES, mixture of catalog #YZ6089, #6093, #6102), and Alexa Fluor 488/555/647-conjugated secondary antibodies (Thermo Fisher Scientific).

    Techniques: Mutagenesis, Derivative Assay, Inhibition, Infection, Expressing, Variant Assay, Construct, Plasmid Preparation, Immunolabeling, Marker, Staining, Two Tailed Test

    Human iPSC-derived neuronal model of Alzheimer’s disease recapitulates key hallmark AD pathologies. a , b Differentiated NAG neurons (12 weeks+) show loss of dendrites (MAP2, green) and cell bodies (CUX2, red) when treated with soluble Aβ species for 7 days ( b ) in comparison to no treatment condition ( a ). c Anti-Aβ antibody co-treatment with soluble Aβ species blocks Aβ-induced cell death. Scale bar 50 μm. d Dose-dependent, progressive cell death as quantified by the percentage of cell body (CUX2) numbers in Aβ-treated normalized to untreated control. e Dose-dependent, progressive dendritic (MAP2) loss as quantified by the percentage of MAP2 area in Aβ-treated normalized to untreated control. f , g Aβ42 treatment induces phosphorylation of Tau (p-Tau 396–404, white) and mislocalization to the cell body. h Anti-Aβ antibody co-treatment with sAβ42s blocks Aβ-induced Tau hyperphosphorylation. Scale bar 50 μm. i Dose-dependent and time course of phosphorylation of Tau at S396/404. Phospho-Tau induced increase at 5 µM Aβ treatment before decrease associated with cell death occurred as quantified by fold p-Tau 396/404 staining in Aβ-treated normalized to untreated control. j , k Aβ42 treatment causes synapse loss in neurons (synapsin, green). l Anti-Aβ antibody co-treatment with sAβ42s blocks synapse loss phenotype. Scale bar = 5 μm. m Dose–response and time course of synapse (synapsin 1/2) loss in Aβ-treated culture normalized to untreated control. n , o sAβ42s treatment induces axon fragmentation (beta-3 tubulin Tuj1, white). p Anti-Aβ antibody co-treatment blocks axon fragmentation. Scale bar = 50 μm. q Dose–response and time course of axon fragmentation as quantified by percentage of the axon (NFL-H) area in Aβ-treated normalized to untreated control. r Anti-Aβ antibody rescues all three markers in a dose-dependent manner and IC50 curves can be drawn and calculated (IC50 curve fitted by Prism software). Data are presented as mean values +/− SEM and n = 4 wells. All scale bars = 50 μm.

    Journal: Nature Communications

    Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

    doi: 10.1038/s41467-021-25344-6

    Figure Lengend Snippet: Human iPSC-derived neuronal model of Alzheimer’s disease recapitulates key hallmark AD pathologies. a , b Differentiated NAG neurons (12 weeks+) show loss of dendrites (MAP2, green) and cell bodies (CUX2, red) when treated with soluble Aβ species for 7 days ( b ) in comparison to no treatment condition ( a ). c Anti-Aβ antibody co-treatment with soluble Aβ species blocks Aβ-induced cell death. Scale bar 50 μm. d Dose-dependent, progressive cell death as quantified by the percentage of cell body (CUX2) numbers in Aβ-treated normalized to untreated control. e Dose-dependent, progressive dendritic (MAP2) loss as quantified by the percentage of MAP2 area in Aβ-treated normalized to untreated control. f , g Aβ42 treatment induces phosphorylation of Tau (p-Tau 396–404, white) and mislocalization to the cell body. h Anti-Aβ antibody co-treatment with sAβ42s blocks Aβ-induced Tau hyperphosphorylation. Scale bar 50 μm. i Dose-dependent and time course of phosphorylation of Tau at S396/404. Phospho-Tau induced increase at 5 µM Aβ treatment before decrease associated with cell death occurred as quantified by fold p-Tau 396/404 staining in Aβ-treated normalized to untreated control. j , k Aβ42 treatment causes synapse loss in neurons (synapsin, green). l Anti-Aβ antibody co-treatment with sAβ42s blocks synapse loss phenotype. Scale bar = 5 μm. m Dose–response and time course of synapse (synapsin 1/2) loss in Aβ-treated culture normalized to untreated control. n , o sAβ42s treatment induces axon fragmentation (beta-3 tubulin Tuj1, white). p Anti-Aβ antibody co-treatment blocks axon fragmentation. Scale bar = 50 μm. q Dose–response and time course of axon fragmentation as quantified by percentage of the axon (NFL-H) area in Aβ-treated normalized to untreated control. r Anti-Aβ antibody rescues all three markers in a dose-dependent manner and IC50 curves can be drawn and calculated (IC50 curve fitted by Prism software). Data are presented as mean values +/− SEM and n = 4 wells. All scale bars = 50 μm.

    Article Snippet: The following primary antibodies were used for immunostaining experiments: Chicken anti-MAP2 (1:2000, GeneTex, GTX85455), Guinea pig anti-Synapsin 1/2 (1:750, Synaptic Systems, #106 004), Mouse anti-Tau HT7 (1:500, Invitrogen, MN1000), Mouse anti-Aβ 6E10 (1:500, Biolegend, #803003), Rabbit anti-CUX2 (1:500, Abcam, ab140329), Rat anti-CTIP2 (1:500, Abcam, ab19465), Guinea pig anti-vGlut2 (1:1000, Synaptic systems, #135304), Mouse anti-Shank (1:200, Millipore N23B/49), Mouse anti-PSD95 (1:200, Millipore, K28/43), Mouse anti-GluR1 (1:200, Millipore), Mouse anti-PanShank (1:200, Millipore, N23B/49), Mouse anti-GluR2 (1:200, Millipore), Mouse anti-PanSAPAP (1:200, Millipore), Mouse anti-NR1 (1:200, Millipore), Rabbit anti-phospho-Tau S396-404 (1:3000, Genentech), Rabbit anti-phospho-Tau S235 (1:1000, ThermoFisher), Mouse anti-β-Tubulin Tuj1 (1:500, Biolegend, #801202), Chick anti-Neurofilament-Heavy Chain (1:3000, Abcam), Rabbit anti-Iba1 (1:1000, Wako Chem, 019-19741), Rabbit anti-TMEM119 (1:500, Abcam, ab209064).

    Techniques: Derivative Assay, Staining, Software

    Dorsal and basal meningeal lymphatic drainage. A LSFM lateral view of OVA-A 555 (magenta) in the posterior head after anti-LYVE1 antibody labeling (green). The animal was sacrificed 45 min after Lb-Sa OVA-A 555 injection. OVA-A 555 is concentrated in brain meninges, within the perivascular spaces along the spinal cord (SC), the cerebellum (Cb) and the cortex (Cx). Outside of the ventral skull border (dashed line), OVA-A 555 is detected in the dcLN. NP: nasopharyngeal cavity (solid line). Dashed rectangles correspond to regions magnified in the indicated panels, MLVs and tracer uptake region are numbered in green (I-IV). Inset in A shows a schematic of dural veins and sinuses (light and dark blue), the light blue veins are the focus of this figure: cav: cavenous sinus, iptgv: interpterygoid emmissary vein, jugv: jugular vein, pfv: posterior facial vein, PSS: petrosquamous sinus, rgv: retroglenoid vein, SS: sigmoid sinus, SSS: superior sagittal sinus, TS: transverse sinus. B-E LSFM horizontal ( B, C ) and sagittal views ( D, E ) of the calvaria, dorsal sinuses are stained with vWF antibody ( B, C ) and MLVs with LYVE antibody ( D , E ). The SSS connects the RCS with the COS ( B , blue arrowheads) which separates in two branches forming the TS ( C ). Note discontinuous OVA-A 555 labeling in the perisinus spaces ( B , C ). LYVE-1+ MLVs line the SSS and TS ( D, I) and contain OVA-A 555 at the TS ( E, II), at the COS and the RCS ( D ), blue arrowheads. F-H LSFM coronal views at the level of the foramen of the jugular vein labeled with the indicated antibodies and OVA-A 555 . vWF stained the SS and the jugv, OVA-A 555 is accumulated around the jugular foramen. ( G ) TUJ1 stained nerves IX, X and XI, which cross the skull through the jugular foramen, OVA-A 555 is close but not associated directly with the nerves. ( H ) LYVE1 + MLVs follow the SS until the jugular vein exits the skull by the jugular foramen. Some MLVs transport OVA-A 555 positive phagocytic cells (III). IX : cranial nerve 9 (Glossopharyngeal), X : cranial nerve 10 (Vagus), X : cranial nerve 11 (Spinal accessory). I-K LSFM coronal views of the petrosquamous sinus exit through the skull.( i ) vWF stained the PSS passing through the petrosquamous sinus foramen (white arrow) to join the pfv via the rgv, OVA-A 555 is accumulated around the jugular foramen. ( J , K ) LYVE1 + MLVs follow the PSS until the rgv and the pfv. Some MLVs show OVA-A 555 + phagocytic cells at the petrosquamous fissure level (blue arrowhead white arrow?). Scale bar: 1000 μm ( A - D ); 500 μm ( E - J ); 250 μm ( K ).

    Journal: bioRxiv

    Article Title: 3D-imaging reveals conserved cerebrospinal fluid drainage via meningeal lymphatic vasculature in mice and humans

    doi: 10.1101/2022.01.13.476230

    Figure Lengend Snippet: Dorsal and basal meningeal lymphatic drainage. A LSFM lateral view of OVA-A 555 (magenta) in the posterior head after anti-LYVE1 antibody labeling (green). The animal was sacrificed 45 min after Lb-Sa OVA-A 555 injection. OVA-A 555 is concentrated in brain meninges, within the perivascular spaces along the spinal cord (SC), the cerebellum (Cb) and the cortex (Cx). Outside of the ventral skull border (dashed line), OVA-A 555 is detected in the dcLN. NP: nasopharyngeal cavity (solid line). Dashed rectangles correspond to regions magnified in the indicated panels, MLVs and tracer uptake region are numbered in green (I-IV). Inset in A shows a schematic of dural veins and sinuses (light and dark blue), the light blue veins are the focus of this figure: cav: cavenous sinus, iptgv: interpterygoid emmissary vein, jugv: jugular vein, pfv: posterior facial vein, PSS: petrosquamous sinus, rgv: retroglenoid vein, SS: sigmoid sinus, SSS: superior sagittal sinus, TS: transverse sinus. B-E LSFM horizontal ( B, C ) and sagittal views ( D, E ) of the calvaria, dorsal sinuses are stained with vWF antibody ( B, C ) and MLVs with LYVE antibody ( D , E ). The SSS connects the RCS with the COS ( B , blue arrowheads) which separates in two branches forming the TS ( C ). Note discontinuous OVA-A 555 labeling in the perisinus spaces ( B , C ). LYVE-1+ MLVs line the SSS and TS ( D, I) and contain OVA-A 555 at the TS ( E, II), at the COS and the RCS ( D ), blue arrowheads. F-H LSFM coronal views at the level of the foramen of the jugular vein labeled with the indicated antibodies and OVA-A 555 . vWF stained the SS and the jugv, OVA-A 555 is accumulated around the jugular foramen. ( G ) TUJ1 stained nerves IX, X and XI, which cross the skull through the jugular foramen, OVA-A 555 is close but not associated directly with the nerves. ( H ) LYVE1 + MLVs follow the SS until the jugular vein exits the skull by the jugular foramen. Some MLVs transport OVA-A 555 positive phagocytic cells (III). IX : cranial nerve 9 (Glossopharyngeal), X : cranial nerve 10 (Vagus), X : cranial nerve 11 (Spinal accessory). I-K LSFM coronal views of the petrosquamous sinus exit through the skull.( i ) vWF stained the PSS passing through the petrosquamous sinus foramen (white arrow) to join the pfv via the rgv, OVA-A 555 is accumulated around the jugular foramen. ( J , K ) LYVE1 + MLVs follow the PSS until the rgv and the pfv. Some MLVs show OVA-A 555 + phagocytic cells at the petrosquamous fissure level (blue arrowhead white arrow?). Scale bar: 1000 μm ( A - D ); 500 μm ( E - J ); 250 μm ( K ).

    Article Snippet: We used the primary antibodies: Goat anti–mouse CD31 (AF3628; R & D Systems, 1:1000), Chicken anti–GFP (GFP10-20; AVES, 1:2000) Goat anti–mouse podocalyxin (AF1556; R & D Systems, 1:1000) Rabbit anti–mouse LYVE-1 (11-034, AngioBio, 1:800), Goat anti– human PROX1 (AF2727; R & D Systems;1:1000), Rabbit anti-mouse TUJ1 (802001, Biolegend; 1:2000), Rabbit anti–human vWF (A0082, Agilent, 1:300).

    Techniques: Antibody Labeling, Injection, Staining, Labeling

    LSFM 3D-images of MLVs associated with the caudal cavernous sinuses in the middle fossa of the skull base. A Schematic of the venous sinuses, veins and internal carotid artery at the base of the brain. Stippled areas correspond to the 3 coronal panels (1-3) shown in ( C , F ) for 1, ( E , H ) for 2 and ( I , J ) for 3. Inset in A : coronal section plane of the head used in B - K . bv: basilar venous plexus, cav: cavernous sinus, ic: internal carotid artery, icav: inter-cavernous sinus, ipets: inferior petrosal sinus, iptgev: interpterygoid emmissary vein, jugv: internal jugular vein, OB: olfactory bulb, pbv: posterior basal vein, pfv: posterior facial vein, spets: superior petrosal sinus. B OVA-A 555 was injected into Th-Lb 45 min before sacrifice and sample was labeled with the indicated antibodies. Blood vessels of the pituitary gland (PG) and dura mater express CD31 and PDLX (blue arrowheads). Tracer deposits (magenta) were observed in the pia mater of the brain, around the cavernous sinus (cav) and the posterior basal vein (pbv). C-E Ventral ( C , D ) and dorsal ( E ) views of a sample stained with vWF to label dural sinuses and veins (blue arrowheads). TS: transverse sinus. Tracer accumulated in peri-venous and -sinusal spaces, especially at confluence points. Dashed line: limit between intracranial and extracranial regions. F-H Lymphatic vasculature (LYVE1 + , green in F , G ) and TUJ1 + cranial nerves (yellow in G ), VI : cranial nerve 6 (Abducens) at coronal levels 1 and 2. MLVs contacted the cavernous perisinus space where tracer accumulated ( F ), and surrounded the foramen of iptgev ( H ). Cranial nerves were devoid of tracer deposits and MLVs. I-L Dural veins (vWF, blue in I ), lymphatic vasculature (LYVE1 + , green in K , L ) and cranial nerves (TUJ1 + , yellow in J ) on coronal ( I-K ) and sagittal ( L ) views at level 3. MLVs contacted the cavernous sinuses ( K , L ) and lymphatic tracer uptake (white in K ) was detected at the intersection of cavernous sinuses with internal carotid arteries in the skull base. Cranial nerves showed neither tracer deposits nor MLVs ( J ). V : cranial nerve 5 (Trigeminal), IV : cranial nerve 4 (Trochlear), III : cranial nerve 3 (Oculomotor). Br: brain, CC: carotid canal, NP: nasopharyngeal cavity. A: anterior, D: dorsal, L: lateral, P: posterior, V: ventral. Scale bar: 500 μm ( B - L ).

    Journal: bioRxiv

    Article Title: 3D-imaging reveals conserved cerebrospinal fluid drainage via meningeal lymphatic vasculature in mice and humans

    doi: 10.1101/2022.01.13.476230

    Figure Lengend Snippet: LSFM 3D-images of MLVs associated with the caudal cavernous sinuses in the middle fossa of the skull base. A Schematic of the venous sinuses, veins and internal carotid artery at the base of the brain. Stippled areas correspond to the 3 coronal panels (1-3) shown in ( C , F ) for 1, ( E , H ) for 2 and ( I , J ) for 3. Inset in A : coronal section plane of the head used in B - K . bv: basilar venous plexus, cav: cavernous sinus, ic: internal carotid artery, icav: inter-cavernous sinus, ipets: inferior petrosal sinus, iptgev: interpterygoid emmissary vein, jugv: internal jugular vein, OB: olfactory bulb, pbv: posterior basal vein, pfv: posterior facial vein, spets: superior petrosal sinus. B OVA-A 555 was injected into Th-Lb 45 min before sacrifice and sample was labeled with the indicated antibodies. Blood vessels of the pituitary gland (PG) and dura mater express CD31 and PDLX (blue arrowheads). Tracer deposits (magenta) were observed in the pia mater of the brain, around the cavernous sinus (cav) and the posterior basal vein (pbv). C-E Ventral ( C , D ) and dorsal ( E ) views of a sample stained with vWF to label dural sinuses and veins (blue arrowheads). TS: transverse sinus. Tracer accumulated in peri-venous and -sinusal spaces, especially at confluence points. Dashed line: limit between intracranial and extracranial regions. F-H Lymphatic vasculature (LYVE1 + , green in F , G ) and TUJ1 + cranial nerves (yellow in G ), VI : cranial nerve 6 (Abducens) at coronal levels 1 and 2. MLVs contacted the cavernous perisinus space where tracer accumulated ( F ), and surrounded the foramen of iptgev ( H ). Cranial nerves were devoid of tracer deposits and MLVs. I-L Dural veins (vWF, blue in I ), lymphatic vasculature (LYVE1 + , green in K , L ) and cranial nerves (TUJ1 + , yellow in J ) on coronal ( I-K ) and sagittal ( L ) views at level 3. MLVs contacted the cavernous sinuses ( K , L ) and lymphatic tracer uptake (white in K ) was detected at the intersection of cavernous sinuses with internal carotid arteries in the skull base. Cranial nerves showed neither tracer deposits nor MLVs ( J ). V : cranial nerve 5 (Trigeminal), IV : cranial nerve 4 (Trochlear), III : cranial nerve 3 (Oculomotor). Br: brain, CC: carotid canal, NP: nasopharyngeal cavity. A: anterior, D: dorsal, L: lateral, P: posterior, V: ventral. Scale bar: 500 μm ( B - L ).

    Article Snippet: We used the primary antibodies: Goat anti–mouse CD31 (AF3628; R & D Systems, 1:1000), Chicken anti–GFP (GFP10-20; AVES, 1:2000) Goat anti–mouse podocalyxin (AF1556; R & D Systems, 1:1000) Rabbit anti–mouse LYVE-1 (11-034, AngioBio, 1:800), Goat anti– human PROX1 (AF2727; R & D Systems;1:1000), Rabbit anti-mouse TUJ1 (802001, Biolegend; 1:2000), Rabbit anti–human vWF (A0082, Agilent, 1:300).

    Techniques: Injection, Labeling, Staining