Journal: Neural regeneration research

Article Title: Sox11b regulates the migration and fate determination of Müller glia-derived progenitors during retina regeneration in zebrafish.

doi: 10.4103/1673-5374.346550

Figure Lengend Snippet: Figure 6 ｜Sox11b regulates the expression of Notch signaling components in injured retinas. (A, B) qPCR revealed the relative expression levels (normalized to 0 days) in the retina at 4 dpi: Notch ligands (A); Notch receptors and the reporter gene her4.1 (B). (C) BrdU immunofluorescence showed that RO4929097-treated MGPCs exhibited a more rounded morphology. Outlines of MGPC nuclei are indicated by white dotted lines. (D) Quantification of MGPC nuclear length/width ratio in the INL. (E) BrdU lineage tracing indicated that Notch inhibition altered the distribution and increased the number of MGPCs, compared with control retinas at 7 dpi. White asterisks indicate sites of stab injury. (F) Quantification of MGPC cell number at the injury site. (G) Quantification of the proportion of BrdU+ cells in each layer. (H) qPCR showed the relative mRNA expression levels (normalized to 0 days) of sox11a and sox11b in PBS- or RO4929097-treated retinas at 4 dpi. Data are expressed as mean ± SEM (n = 3). Values were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. BrdU: 5-Bromo-2′-deoxyuridine; dpi: days post injury; GCL: ganglion cell layer; INL: inner nuclear layer; MO; morpholino; MGPC: Müller glia-derived progenitor cells; ns: not significant; ONL: outer nuclear layer; PBS: phosphate-buffered saline.

Article Snippet: For Notch inhibition, 1 μL of 250 μM of RO4929097 (MedChemExpress, Monmouth Junction, NJ, USA) was intravitreally injected through the front of the eye from 2 to 4 dpi, twice daily.

Techniques: Expressing, Immunofluorescence, Inhibition, Control, Derivative Assay, Saline

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Notch signaling inactivation by small molecule γ-secretase inhibitors restores the multiciliated cell population in the airway epithelium

doi: 10.1152/ajplung.00382.2022

Figure Lengend Snippet: GSI treatment induces multiciliated cell formation. A : primary human nasal epithelial cells were treated during differentiation (ALI + 0 to +21 days) with a range of concentrations of LY450139 and labeled at ALI + 21 days with anti-acetylated α-Tubulin (green) and ECAD (red) antibodies to mark cilia and epithelial junctions. High concentration (10 µM) of LY450139 disrupted the epithelial junctions, but lower concentrations lead to a dose-dependent increase in multiciliated cell numbers similar to DAPT. Scale bar, 50 µm. B : quantitation of data shown in A . One-way ANOVA, ns, not significant, * P < 0.01, ** P < 0.001. ALI, air-liquid interface; GSI, γ-secretase inhibitor.

Article Snippet: GSI compounds (DAPT, LY450139, PF-03084014, RO-4929097, and MK-0752) were obtained from Selleckchem and reconstituted in DMSO then diluted in culture medium (HNEC treatment) or saline (mouse IP injection).

Techniques: Labeling, Concentration Assay, Quantitation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Notch signaling inactivation by small molecule γ-secretase inhibitors restores the multiciliated cell population in the airway epithelium

doi: 10.1152/ajplung.00382.2022

Figure Lengend Snippet: GSI treatment induces multiciliated cell formation in differentiating and mature airway epithelia. A : primary human nasal epithelial cells were treated with DAPT and LY450139 during pre-ALI only (ALI-5 to -1 day = proliferation), during ALI only (ALI + 0 to +21 days = differentiation) or continuously during pre-ALI and ALI (ALI-5 days to +21 days = all times) and labeled at ALI + 21 days with anti-acetylated α-Tubulin (green) and ECAD (red) antibodies. Only GSI treatment during differentiation and all times increased multiciliated cell numbers. GSI treatment during pre-ALI (proliferation only and all times treatments) had no detrimental effect on multiciliated cells or overall epithelial structure. Scale bar, 50 µm. B : quantitation of data shown in A . One-way ANOVA, ns, not significant; * P < 0.0001. C : mature (ALI + 30 days) primary human nasal epithelial cells were treated with DAPT and LY450139 for one (ALI + 30 to +37 days) or 2 wk (ALI + 30 to +44 days), then labeled with anti-acetylated α-Tubulin (green) and ECAD (red) antibodies. GSI treatment induces the formation of additional multiciliated cells in mature cultures, while untreated cultures do not form any more multiciliated cells. Scale bar, 50 µm. D : quantitation of data shown in C . One-way ANOVA, * P < 0.0001. E : primary human nasal epithelial cells were treated with DAPT and LY450139 during differentiation only (ALI + 0 to +21 days) from either the apical or basal surface, then labeled at ALI + 21 days with anti-acetylated α-Tubulin (green) and ECAD (red) antibodies. GSI treatment induces multiciliated cell formation via both apical and basal application. Apical treatment abrogates the air-liquid interface, which results in the poor epithelial structure and multiciliated cell differentiation in the untreated cultures, which is partially rescued by GSI treatment. Scale bar, 50 µm. F : quantitation of data shown in E . One-way ANOVA, * P < 0.01, ** P <0.001. ALI, air-liquid interface; GSI, γ-secretase inhibitor.

Article Snippet: GSI compounds (DAPT, LY450139, PF-03084014, RO-4929097, and MK-0752) were obtained from Selleckchem and reconstituted in DMSO then diluted in culture medium (HNEC treatment) or saline (mouse IP injection).

Techniques: Labeling, Quantitation Assay, Cell Differentiation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Notch signaling inactivation by small molecule γ-secretase inhibitors restores the multiciliated cell population in the airway epithelium

doi: 10.1152/ajplung.00382.2022

Figure Lengend Snippet: GSI treatment induces multiciliated cell formation in vivo. A : adult Foxj1-EGFP mice were treated with 10 mg/kg DAPT, 1 mg/kg or 0.1 mg/kg LY450139 or vehicle control twice daily for 3 days, then on day 7 multiciliated cell number was quantitated in airway cross sections based on GFP fluorescence, which shows that 1 mg/kg LY450139 increased multiciliated cell number. Treatment with 0.1 mg/kg LY450139 showed a similar trend but did not reach significance ( P = 0.09). n = 4 mice were treated per category. Scale bar, 100 µm. B : quantitation of data shown in A . One-way ANOVA, ns, not significant, * P < 0.05. C : adult Foxj1-EGFP mice were treated with 1 mg/kg LY450139 or vehicle control once daily for five consecutive days for 3 wk, then on day 30 multiciliated cell number was quantitated, which shows that 1 mg/kg LY450139 increased multiciliated cell number. n = 8 mice were treated per category. Scale bar, 100 µm. D : quantitation of data shown in C . T test, * P < 0.01. E : mouse body weights over the course of the experiment in C – D depicted as a proportion of the day 1 weight for individual mice ( left ) and as the average of the absolute weights ( right ), T test, ns, not significant. ALI, air-liquid interface; GSI, γ-secretase inhibitor.

Article Snippet: GSI compounds (DAPT, LY450139, PF-03084014, RO-4929097, and MK-0752) were obtained from Selleckchem and reconstituted in DMSO then diluted in culture medium (HNEC treatment) or saline (mouse IP injection).

Techniques: In Vivo, Control, Fluorescence, Quantitation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Notch signaling inactivation by small molecule γ-secretase inhibitors restores the multiciliated cell population in the airway epithelium

doi: 10.1152/ajplung.00382.2022

Figure Lengend Snippet: GSI treatment induces multiciliated cell formation in epithelia with mucous cell hyperplasia. A : primary human nasal epithelial cells were treated with recombinant human IL-13 during early epithelial differentiation (ALI + 7 to +14 days), which induced mucous cell formation at the expense of multiciliated cell differentiation as assessed by anti-acetylated α-Tubulin (green), MUC5AC (red) and ECAD (blue) antibodies. These cultures were then treated with DAPT or LY450139 for an additional week (ALI + 14 to +21 days), then assessed at ALI + 21 days. Although the untreated cells maintained the remodeled phenotype, GSI treatment induced multiciliated cell formation to levels comparable to healthy epithelia. Arrows point to acetylated α-Tubulin and MUC5AC double positive cells in GSI treated cultures. Scale bar, 50 µm. B : quantitation of data shown in A . One-way ANOVA, * P < 0.0001. ALI, air-liquid interface; GSI, γ-secretase inhibitor.

Article Snippet: GSI compounds (DAPT, LY450139, PF-03084014, RO-4929097, and MK-0752) were obtained from Selleckchem and reconstituted in DMSO then diluted in culture medium (HNEC treatment) or saline (mouse IP injection).

Techniques: Recombinant, Cell Differentiation, Quantitation Assay

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure1. Notch-inhibitedretinasexhibitincreasedMu¨llergliaactivationandproliferationafterlightdamage.a,AdultTg(gfap:EGFP)nt11zebrafish(green,row1)werelight-damagedfor24h andthenintravitreallyinjectedwitheither100MRO4929097or1%DMSO.After36hofconstantlight,RO4929097-treatedretinas(column1)exhibitedincreasednumbersofPCNA-positive(red, row 2) and Ascl1a-positive (blue, row 3) Mu¨ller glia relative to DMSO-treated retinas (column 2). Arrows (column 1) indicate Mu¨ller glia nuclei double-positive for Ascl1a and PCNA. b, Histogram depicting no change in the number of INL Mu¨ller glia but a significant increase in the number of PCNA-positive and Ascl1a-positive INL cells in the RO4929097-treated light-damaged Tg(gfap: EGFP)nt11 retinas (n 8). c, Adult Tg(gfap:EGFP)nt11 zebrafish (green, row 1) were intraperitoneally injected with EdU at 36 h of constant light, intravitreally injected with either 100 M RO4929097(column1)or1%DMSO(column2)at48and60h,pulsedintraperitoneallywithBrdUat63and66h,andimmunolabeledat72hoflightforEGFP(green,row1),BrdU(red,row2),and EdU(blue,row3).TheRO4929097-treatedretinas(column1)containedincreasednumbersofBrdU-positiveINLcellclustersthatwereEdU-negative(arrows)relativetotheDMSOcontrol(column 2).d,HistogramdepictingnochangeineitherthenumberofEGFP-positiveorEdU-positiveMu¨llergliaat72hoflight-damageinDMSOorRO492097-treatedretinas.Thenumberofnewlyactivated BrdU-positive/EdU-negativecellclusters,however,weresignificantlyincreasedby72hinRO492097-treatedretinasrelativetotheDMSOcontrolretinas(n8).GCL,Ganglioncelllayer;INL,inner nuclear layer; ONL, outer nuclear layer; PCNA, proliferating cellular nuclear antigen. Scale bars, 25 m. Student’s t test, *p 0.05. **p 0.01.

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Injection

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure 2. Inhibition of Notch signaling induces Mu¨ller glia proliferation in the absence of retinal damage. a, Intraperitoneal injection of 1 mM RO4929097 (bottom row) into AB zebrafish demonstrates an increased number of PCNA-positive INL cells (green, arrows) relative to the 1% DMSO-injected controls (top row). b, RO4929097 treatment results in a significant increase in the numberofPCNA-positiveINLcellsrelativetotheDMSOcontrolat2,3,and4d(n7).c,AdultalbinozebrafishwereintraperitoneallyinjectedwithRO4929097(alb RO4929097 HS,column1), andTg(hsp70l:Gal4);Tg(UAS:myc-notch1a-intra)transgeniczebrafishwereintraperitoneallyinjectedwitheitherRO4929097(Tg RO4929097 HS,column3)orDMSO(Tg DMSO HS,column 2)andheat-shockedfor3d.NICD-Myc(red)wasexpressedintheheat-shockedTg(hsp70l:Gal4);Tg(UAS:myc-notch1a-intra)zebrafishandnotintheheat-shockedalbinofish.ExpressionofNICD-Mycreducedthe number of PCNA-positive INL cells (green) that were induced by RO4929097. NICD-Myc-positive Mu¨ller glia (arrows) did not express PCNA. d, Tg(hsp70l:Gal4);Tg(UAS:myc-notch1a-intra) zebrafish were intraperitoneallyinjectedwithRO4929097andheat-shockedfor3d.TheNICD-Mycfusionproteinwasinducedintheglutaminesynthetase-positiveMu¨llerglia(arrows).e,notch1a,notch1b,andnotch3mRNA expressionwaslocalizedtothebasalINLoftheundamagedretinabyinsituhybridization.f,qRT-PCRrevealedthatRO4929097inhibitionofNotchsignalingsignificantlyreducedthetranscriptlevelsofthe Notch-specifictargetsher4andher6at2and3dandincreasedexpressionoftheactivatedMu¨llergliamarkersascl1aandstat3(n4).g,TissuesectioninsituhybridizationverifiedadownregulationoftheNotch downstreamtarget,her6,by3dofRO4929097treatment.GCL,Ganglioncelllayer;INL,innernuclearlayer;ONL,outernuclearlayer;PCNA,proliferatingcellularnuclearantigen.Scalebars,25m.ToPro3(a,e, g)andDAPIstaining(c,d)wereusedtodistinguishthethreenuclearlayers.*p0.05(Student’sttest).**p0.01(Student’sttest).***p0.001(Student’sttest).

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Inhibition, Injection

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure 4. Inhibiting Notch signaling in the undamaged retina induces Ascl1a-dependent Mu¨ller glia proliferation. a, Undamaged adult Tg(gfap:EGFP)nt11 zebrafish (green, row 1) were intraperitoneallyinjectedwitheither1mMRO4929097or10%DMSO.Confocalimagesof1mMRO4929097-treatedretinasimmunolabeledforPCNA(red,row2)orAscl1a(blue,row3)at3and4d (columns 1 and 2, respectively) show increased numbers of both PCNA-positive and Ascl1a-positive Mu¨ller glia (merge) relative to the DMSO-injected controls (column 3). Additionally, the PCNA-positive cells colabeled (arrow) with the Ascl1a-positive activated Mu¨ller glia. b, Tg(gfap:EGFP)nt11 were either intraperitoneally injected with 1 mM RO4929097 (column 1) or intravitreally injectedandelectroporatedwithascl1aMO(column2)followedbyintraperitonealinjectionwith1mMRO4929097for3d.KnockdownofAscl1aexpressiondecreasedthenumberofPCNA-positive (red,row1)andAscl1a-expressing(bluerow2)cells.AlthoughAscl1alevelswereclearlydecreasedinthemorphantretina,averysmallnumberofAscl1a-positive(singlearrow)andAscl1a-negative (double arrow) PCNA-expressing cells remained. c, Histogram depicting a significant decrease in the number of RO4929097-induced PCNA-positive cells in the INL following ascl1a morpholino knockdown (n 8). GCL, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PCNA, proliferating cellular nuclear antigen; MO, morpholino. Scale bars, 25 m. **p 0.01 (Student’s t test).

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Injection, Expressing, Knockdown

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure5. InhibitingNotchsignalingintheundamagedretinainducesStat3expressioninproliferatingMu¨llerglia.UndamagedTg(gfap:EGFP)nt11zebrafishwereintraperitoneallyinjectedwith either 1 mM RO4929097 or 10% DMSO for 3 or 4 d and immunolabeled for EGFP (a–c, green), PCNA (d–f, red), and Stat3 (g–i, blue). 1 mM RO4929097-treated Tg(gfap:EGFP)nt11 retinas at 3 d (column1)and4d(column2)showedincreasednumbersofPCNA-positive(d,e,red)andStat3-positive(g,h,blue)cellsthatcolabeledEGFP-positiveMu¨llerglia(j,k,l,mergearrows)relativeto the DMSO-injected controls (column 3). GCL, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PCNA, proliferating cellular nuclear antigen. Scale bars, 25 m.

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Immunolabeling, Injection

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure6. Jak-mediatedStat3phosphorylation/activationisnecessaryfortheNotch-inhibitedundamagedretinatoproliferateindependentofTNF.a,albinofishwereintraperitoneallyinjected withDMSO,RO4929097andDMSO,RO4929097andruxolitinib,orRO4929097andStatticfor3d.WhereasRO4929097andDMSOwassufficienttoinducePCNA-positiveINLcells,blockingtheStat3 signaling pathway with either ruxolitinib or Stattic inhibited the RO4929097-induced proliferation. b, Histogram depicting that the number of PCNA-positive INL cells in RO4929097-treated undamaged retinas was significantly reduced by cotreating with either ruxolitinib or Stattic (n 8). c, d, Total protein lysates from DMSO, RO4929097 and DMSO, RO4929097 and ruxolitinib- treated retinas confirmed increased total Stat3 (c) and p-Stat3–708 (d) protein levels in the RO4929097-treated undamaged retinas and near control levels in the RO4929097 and ruxolitinib- cotreatedretinas.-Actinwasusedasaloadingcontrol.e,Confocalimagesof36hlight-damagedretina(column1)thatwasimmunolabeledforTNF(green)andPCNA(red)showrobustTNF expressioninPCNA-positiveINLcells.Undamagedalbinozebrafishwereintraperitoneallyinjectedwitheither1mMRO4929097or10%DMSOvehiclecontrolforeither2or3d(columns2,3,and4). TheRO4929097andDMSOcontrolretinalsectionswereimmunolabeledforTNF(green,row1)andmergedwithPCNA(red,row2)andthenuclearmarkerToPro3(blue,row2).AlthoughTNF expression was detected in both the outer plexiform layer and nerve fiber layer (arrow and arrowhead, respectively), there was no detectable TNF expression in the INL or ONL of either theRO4929097orDMSO-treatedretinas.ThisdemonstratesthatRO4929097-inducedPCNAexpressionintheINLisnottheresultofincreasedTNFexpressionintheINLMu¨llerglia.GCL, Ganglioncelllayer;INL,innernuclearlayer;ONL,outernuclearlayer;PCNA,proliferatingcellularnuclearantigen;Rux,ruxolitinib.Scalebars,25m.***p0.001(one-wayANOVAand Tukey’s post hoc test).

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Control, Expressing

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure7. TheproliferatingneuronalprogenitorsproducedbyinhibitingNotchsignalingintheundamagedretinadonotdifferentiateandundergocelldeath.UndamagedalbinoTg(atoh7:GFP) zebrafish (a) and albino Tg[gfap:EGFP]nt11 transgenic zebrafish (b, c) were intraperitoneally injected with either 1 mM RO4929097 or 10% DMSO every 12 h for 4 d, EdU was coinjected at 1.5, 2.5, and3.5d,andretinaswerecollectedat5,7,9,or11d.a,ConfocalimageofTg(atoh7:GFP)(green,column1)retinasthatwerelight-damagedfor72hdisplayedrobustPCNAexpression(red,column 1) in the GFP-positive INL clusters. RO4929097-treated Tg(atoh7:GFP) zebrafish (columns 2–4) displayed large numbers of EdU-positive INL cells (blue, lower row) relative to the DMSO control (column 5). Although limited, there was also an increase in the number of GFP-positive INL cells that colabel with EdU-positive INL cells (arrow) in the RO4929097-treated retinas relative to the DMSO-treatedretinas.b,c,ConfocalimagesofTg[gfap:EGFP]nt11transgeniczebrafish(whichexpressEGFPintheMu¨llergliafromthegfappromoter)treatedwitheither1mMRO4929097(column 1)or10%DMSO(column2)for7d(b)and11d(c)andlabeledwithanti-GFPtoidentifytheMu¨llerglia(green,row1)andTUNEL(red,row2).ArrowsindicateMu¨llergliathatareTUNEL-positive. d, Histogram depicting an increased number of TUNEL-positive INL cells at both 7 and 11 d in RO4929097-injected retinas compared with 10% DMSO (n 5). GCL, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PCNA, proliferating cellular nuclear antigen. Scale bars, 25 m. Student’s t test, *p 0.05.

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Transgenic Assay, Injection, Control, TUNEL Assay

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure8. InhibitionofNotchsignalingincombinationwithexogenousTNFactsynergisticallytoinducerobustMu¨llergliaproliferation.a,AdultTg(gfap:EGFP)nt11transgeniczebrafishwere intraperitoneallyinjectedwitheither1mMRO4929097or10%DMSOincombinationwithanintravitrealinjectionofeitherpurifiedrecombinantTNForpurifiedcontrolbacteriallysate(CL)for2–4 d.TheTNF/DMSO,RO4929097/CL,andTNF/RO4929097coinjectedretinaspossessedsignifcantlymorePCNA-positivecells(red)at2,3,and4dofinjectionsrelativetotheCL/DMSOcoinjected control. The fourth column (4 d PCNA) contains higher-magnification images of PCNA labeling to highlight the single INL cells in the TNF/DMSO coinjected retina, the single and double cells in the RO4929097/CL coinjected retina, and the columns containing several PCNA-positive cells in the TNF/RO4929097 coinjected retina. b, Histogram depicting a significant increase in the number of PCNA-positive INL cells in the TNF/DMSO, RO4929097/CL, and RO4929097/TNF-coinjected retinas compared with the CL/DMSO coinjected control (n 6). c, Histogram showing a significant increase in the number of dorsal and ventral PCNA-positive Mu¨ller glia in the TNF/DMSO, RO4929097/CL, and RO4929097/TNF-coinjected retinas compared with the CL/DMSO coinjected control (n 6), whereas the total number of Mu¨ller glia remained unchanged in all four treatment groups. GCL, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PCNA, proliferating cellular nuclear antigen. Scale bars, 25 m. **p 0.01 (two-way ANOVA and Tukey’s post hoc test). ***p 0.001 (two-way ANOVA and Tukey’s post hoc test).

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Control, Labeling

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure 9. Robust proliferation stimulated by coinjection of RO4929097 and TNF stimulates neuronal progenitors to commit to a neuronal fate. Tg(atoh7:GFP) or Tg(nrd:GFP) zebrafish were coinjected with TNF/DMSO, RO4929097/CL, and RO4929097/TNF for 4 d. a, The Tg(atoh7:GFP) zebrafish were collected at 4 and 5 d and immunolabeled for PCNA (red). The RO4929097/TNF coinjected retinas had significantly more GFP-positive cell clusters that robustly colabeled with PCNA than either of the other two treatments. b, Histogram depicting the increased number of GFP-positiveINLclusterspresentintheRO4929097/TNFcotreatedretinasrelativetotheothertwotreatments(n7).c,TheTg(nrd:egfp)zebrafishwerecollectedat5dandimmunolabeledfor PCNA(red).TheRO4929097/TNFcoinjectedretinashadmorediffuseGFP-positivecells(representingcommittedneuronalprogenitorcells)thatrobustlycolabeledwithPCNA(arrows)thaneither oftheothertwotreatments.GCL,Ganglioncelllayer;INL,innernuclearlayer;ONL,outernuclearlayer;PCNA,proliferatingcellularnuclearantigen.Scalebars,25m.**p0.01(two-wayANOVA and Tukey’s post hoc test). ***p 0.001 (two-way ANOVA and Tukey’s post hoc test).

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Immunolabeling

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure 10. Coinjection of RO4929097 and TNF reduces neuronal progenitor cell death. Tg(atoh7:GFP) zebrafish were coinjected with TNF and RO4929097 and labeled with EdU. After7d(a,c,e,g)and11d(b,d,f,h),theretinaswereimmunolabeledforGFP(a,b,g,h),EdU (c,d,g,h),andTUNEL(e–h).Themergedimagesofthethreedifferentlabelsareshown(g, h). After 7 d, the retina possessed EdU-labeled cells that were GFP-positive (arrows). No TUNEL-positive cells were detected in the retinas at either day. GCL, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 25 m.

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques: Labeling, TUNEL Assay

Journal: Journal of Neuroscience

Article Title: Repressing Notch Signaling and Expressing TNF Are Sufficient to Mimic Retinal Regeneration by Inducing Muller Glial Proliferation to Generate Committed Progenitor Cells

doi: 10.1523/jneurosci.0498-14.2014

Figure Lengend Snippet: Figure 11. Coinjection of RO4929097 and TNF is sufficient to induce neuronal progenitor cells to differentiate into most types of retinalneurons.Wild-type,Tg(nrd:egfp),orTg(rho:Eco.NfsB-EGFP)nt19zebrafishwerecoinjectedwithTNFandRO4929097andlabeled withEdU.After11d,theretinaswereimmunolabeledforEdU(a,c,d,f,g,i,j,l,m,o,p,r),Zpr-1(b,c),EGFP(e,f,h,i),HuC/D(k,l),PKC (n,o),pERK(q,r),andDAPI(c,f,i,l,o,r).EdU-labeledcone(a–c)androd(d–i)aremarkedwitharrowheads.EdU-labeledbipolarcells (g–i),amacrinecells(j–l),andMu¨llerglia(p–r)areindicatedwitharrows.TherearenodetectableEdU-labeledganglioncells(j–l)or on-bipolarcells(m–o).GCL,Ganglioncelllayer;INL,innernuclearlayer;ONL,outernuclearlayer.Scalebars,25m.

Article Snippet: A small incision was made in the cornea adjacent to the iris of each eye with a sapphire blade scalpel (World Precision Instruments) and 1 l of 1% DMSO (as a vehicle control), 250 M Compound E (Santa Cruz Biotechnology), or 100 M RO4929097 (2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d] azepin-7-yl)-N -(2,2,3,3,3-pentafluoro-propyl)-malonamide; SelleckBio) was intravitreally injected with a blunt-ended 30 gauge syringe (Hamilton Company) every 12 h (Nelson et al., 2012) until either 36 or 72 h after starting the light treatment.

Techniques:

Journal: Cancer research

Article Title: Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

doi: 10.1158/0008-5472.CAN-09-1843

Figure Lengend Snippet: RO4929097 chemical structure and in vitro Aβ40 and Notch potency. A, RO4929097 was derived from a dibenzazepinone lead. B, RO4929097 was assayed in a cell-free γ-secretase enzyme assay and two cellular assays using engineered cell lines to monitor Aβ40 and Notch processing.

Article Snippet: The potency of RO4929097 in cell-free and cellular assays was in the low nanomolar range with >100-fold selectivity observed with respect to 75 other proteins of various types including receptors, ion channels, and enzymes (CEREP panel; data not shown).

Techniques: In Vitro, Derivative Assay, Enzymatic Assay

Journal: Cancer research

Article Title: Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

doi: 10.1158/0008-5472.CAN-09-1843

Figure Lengend Snippet: RO4929097 inhibits the production of ICN, reducing the expression of the downstream Notch target Hes1, producing a less transformed morphology in A549 cells. A, RT-PCR analysis was used to monitor Hes1 mRNA expression. B, Western blot analysis was used to monitor the protein expression of ICN and Hes1. C, tissue culture pictures showing a less transformed morphology. D, RO4929097 reduces the size of MDA-MB-468 soft agar colonies. Pictures were taken 3 weeks after start of dosing. Compound was added twice weekly.

Article Snippet: The potency of RO4929097 in cell-free and cellular assays was in the low nanomolar range with >100-fold selectivity observed with respect to 75 other proteins of various types including receptors, ion channels, and enzymes (CEREP panel; data not shown).

Techniques: Expressing, Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cancer research

Article Title: Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

doi: 10.1158/0008-5472.CAN-09-1843

Figure Lengend Snippet: In vivo efficacy of RO4929097 in the A549 xenograft model. A, nude mice bearing A549 s.c. tumors were dosed orally following the indicated schedule. Arrow, length of treatment. B, animals from the 60 mg/kg group were monitored after cessation of treatment for 32 d and then redosed for 7 d. C, animals were dosed on a daily 21-day schedule at the indicated concentrations and then monitored after cessation of treatment. D, summary of breadth of in vivo efficacy. TGI, tumor growth inhibition; bid, twice daily; qd, once daily.

Article Snippet: The potency of RO4929097 in cell-free and cellular assays was in the low nanomolar range with >100-fold selectivity observed with respect to 75 other proteins of various types including receptors, ion channels, and enzymes (CEREP panel; data not shown).

Techniques: In Vivo, Inhibition

Journal: Cancer research

Article Title: Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

doi: 10.1158/0008-5472.CAN-09-1843

Figure Lengend Snippet: RO4929097-treated A549 tumors have increased areas of necrosis with relative increase in extracellular matrix (ECM). A, representative H&E-stained tumor tissue sections from vehicle and 10 mg/kg/d × 21 d RO4929097-treated groups. B, Western blot analysis of xenograft protein was done. C, angiogenesis-specific RT-PCR array showing down-regulation of several angiogenic genes in the sensitive A549 xenograft model compared with the insensitive H460a model.

Article Snippet: The potency of RO4929097 in cell-free and cellular assays was in the low nanomolar range with >100-fold selectivity observed with respect to 75 other proteins of various types including receptors, ion channels, and enzymes (CEREP panel; data not shown).

Techniques: Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer research

Article Title: Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

doi: 10.1158/0008-5472.CAN-09-1843

Figure Lengend Snippet: Comparative microarray analysis. A, heat map of human gene expression changes induced by RO4929097 at 6 and 24 h in A549 and H460 NSCLC-derived cell lines as monitored (Affymetrix U133 Plus 2 arrays). Red and green, genes up-regulated and down-regulated at least 1.5-fold after RO4929097 treatment (P < 0.001, ANOVA, compared with untreated controls), respectively. Boxes list mRNAs that were altered at both 6 and 24 h. B, heat map of genes that are differentially expressed in both RO4929097-treated A549 cells (versus untreated) and well-differentiated non–small cell lung adenocarcinomas (versus poorly differentiated) identified by Gene Set Enrichment Analysis using the NextBio software.

Article Snippet: The potency of RO4929097 in cell-free and cellular assays was in the low nanomolar range with >100-fold selectivity observed with respect to 75 other proteins of various types including receptors, ion channels, and enzymes (CEREP panel; data not shown).

Techniques: Microarray, Expressing, Derivative Assay, Software

Journal: PeerJ

Article Title: Curcumol alleviates liver fibrosis by inducing endoplasmic reticulum stress-mediated necroptosis of hepatic stellate cells through Sirt1/NICD pathway

doi: 10.7717/peerj.13376

Figure Lengend Snippet: (A) The protein abundance of NICD was analyzed. (B) Immunofluorescence staining of NICD (green). (C) The protein abundances of NICD after Sirt1 knockdown was investigated. (D) The inhibitory effect of γ-secretase inhibitor Ro4929097 (1 µM) on NICD was verified. (E) Cell viability was assayed by MTT. (F) Western blot was used to test the effect of Ro4929097 on RIP1 and RIP3 in HSCs. (G) CO-IP assay detected the interaction between Sirt1 and NICD. (H) The acetylation level of NICD was assayed by IP assay. Data are expressed as mean ± SD ( n = 3); * P < 0.05, ** P < 0.01, and *** P < 0.001 vs DMSO. $ P < 0.05, and $$$ P < 0.001 vs curcumol. ### P < 0.001 vs Cur+siCtrl. @ P < 0.05, @@@ P < 0.001 vs Cur+siSirt1.

Article Snippet: RO4929097 was provided by APExBIO (Houston, TX, USA).

Techniques: Quantitative Proteomics, Immunofluorescence, Staining, Knockdown, Western Blot, Co-Immunoprecipitation Assay