Journal: Cell and Tissue Research

Article Title: The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea

doi: 10.1007/s00441-021-03493-w

Figure Lengend Snippet: Co-treatment with DAPT and QS11 promotes SC proliferation and HC regeneration in the cochlea in vitro in an age-dependent manner. The P0–P1 cochleae were cultured with 10 μM QS11 a or 5 μM DAPT b or with both QS11 and DAPT c for 3 days. There were significantly greater numbers of EdU + /Sox2 + cells and EdU + /Myo7a + cells in the apex of the cochlea compared to the DAPT-only group and the QS11-only group. The EdU + cells in the DAPT + QS11 co-treated cochleae were seen throughout the sensory cell region in the apical turn. Scale bar = 20 μm. A control cochlea treated with vehicle is shown in d (Scale bar = 50 μm). The histograms show the differences in the numbers of EdU + cells e and Myo7a + cells f between these groups. The cells were counted per 100-μm length along the cochlea. The numbers of EdU + cells were counted between the outer HCs and the inner HCs. (* p < 0.05, ** p < 0.01, *** p < 0.001). The P4 cochleae were cultured with 10 μM QS11 g – g ’” or 5 μM DAPT h – h ’” or with both QS11 and DAPT i – i ’” for 3 days. The number of EdU + and Myo7a + cells was greater than in the DAPT-only and QS11-only groups, while the numbers of Myo7a + cells and EdU + cells in the sensory area were fewer compared to P0–P1 cochleae with the same treatment. Scale bar = 20 μm. The histograms show the differences in the numbers of Myo7a + cells j and EdU + cells k in the apical turn of the cochlea between the P0–P1 mice and the P4 mice for the different treatment groups. The cells were counted per 100-μm length along the cochlea. The numbers of EdU + cells were counted between the outer HCs and the inner HCs. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cultures were exposed to culture medium with 10% FBS for about 2 h and then given the following treatments: DAPT (γ-secretase inhibitor IX, N -[ N -(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butylester, EMD, Gibbstown, NJ, USA); QS11 ((2S)-2-[2-(indan-5-yloxy)-9-(1,1′-biphenyl-4-yl) methyl]-9H–purin-6-ylamino)-3-phenyl-propan-1-ol, Tocris Biosciences, Ellisville, Missouri, USA), which modulates ARF-GTP levels and synergizes with the Wnt/β-catenin signaling pathway to upregulate β-catenin nuclear translocation; and recombinant SHH protein (R & D Systems, Minneapolis, MN, USA), which activates the SHH signaling pathway.

Techniques: In Vitro, Cell Culture, Control

Journal: Cell and Tissue Research

Article Title: The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea

doi: 10.1007/s00441-021-03493-w

Figure Lengend Snippet: Co-treatment with DAPT and QS11 promotes SC proliferation and HC regeneration in neomycin-damaged cochleae in vitro. The P0–P1 cochleae were cultured with 1.0 mM neomycin for 16 h, then treated with either media only a – a ”, 5 μM DAPT b – b ”, 10 μM QS11 c – c ”, or both d – d ” for another 7 days. The number of Myo7a + cells was decreased in the apical, middle, and basal turns of the cochlea, especially in the basal turn and middle turns in the control and QS11-only groups. However, the numbers of EdU + /Sox2 + cells and EdU + /Myo7a + cells were increased in the DAPT-only and DAPT + QS11 groups, especially in the apical turn of the cochlea. In addition, there were many more EdU + cells in the sensory area of the cochlea than in the DAPT-only group, especially in the apical turn of the cochlea (scale bar = 20 μm). The histograms show the differences in the numbers of EdU + cells e and Myo7a + cells f in the different turns of the cochlea. The cells were counted per 100-µm length along the cochlea, which was measured between the outer HCs and the inner HCs (** p < 0.01, *** p < 0.001). The P0–P1 Atoh1-eGFP transgenic mouse cochleae were cultured with 2.0 mM neomycin for 16 h then treated with either media only g – g ”, 5 μM DAPT h – h ”, 10 μM QS11 i – i ”, or both j – j ” for another 7 days. Myo7a + cells were clearly lost in the basal turn and the middle turn of the cochlea in the control and QS11-only groups, and there were no EdU + /Sox2 + cells or EdU + /Myo7a + cells in the sensory domain in these two groups. However, there were some EdU + /Sox2 + cells and EdU + /Myo7a + cells in the sensory area of the cochleae in the DAPT-only and the combination groups, with more cells seen in the combination group compared to the DAPT-only group, especially in the apical turn of the cochlea. The P0–P1 Atoh1-eGFP mouse cochleae were cultured only with EdU, and there were no obvious EdU + /Sox2 + cells or EdU + /Myo7a + cells in the sensory area k . The histograms show the differences in the numbers of EdU + cells l and Myo7a + cells m in different turns of the cochlea. The cells were counted per 100-µm length along the cochlea, which was measured between the outer hair cells and the inner hair cells (* p < 0.05, ** p < 0.01)

Article Snippet: Cultures were exposed to culture medium with 10% FBS for about 2 h and then given the following treatments: DAPT (γ-secretase inhibitor IX, N -[ N -(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butylester, EMD, Gibbstown, NJ, USA); QS11 ((2S)-2-[2-(indan-5-yloxy)-9-(1,1′-biphenyl-4-yl) methyl]-9H–purin-6-ylamino)-3-phenyl-propan-1-ol, Tocris Biosciences, Ellisville, Missouri, USA), which modulates ARF-GTP levels and synergizes with the Wnt/β-catenin signaling pathway to upregulate β-catenin nuclear translocation; and recombinant SHH protein (R & D Systems, Minneapolis, MN, USA), which activates the SHH signaling pathway.

Techniques: In Vitro, Cell Culture, Control, Transgenic Assay

Journal: Cell and Tissue Research

Article Title: The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea

doi: 10.1007/s00441-021-03493-w

Figure Lengend Snippet: The role of SHH with DAPT and QS11 in the intact inner ear in vitro. Neonatal P0–P1 cochleae were treated with either 200 ng/ml SHH a – a ”, d – d ”, i – i ’”, 5 μM DAPT, or 10 μM QS11 b – b ”, e – e ”, j – j ’” or the combination of all three c – c ”, f – f ”, k – k ’” for 3 days. There were EdU + /Sox2 + cells and EdU + /Myo7a + cells in the sensory area of the cochlea. The numbers of EdU + cells in the DAPT + QS11 + SHH group were much greater than those in the DAPT + QS11 group a – a ”, b – b ”, c – c ”, especially in the apical turn of the cochlea d – d ”, e – e ”, f – f ”. There were many more EdU + /Sox2 + cells in the GER of the cochlea in the DAPT + QS11 + SHH group compared to the other groups i – i ’”, j – j ’”, k – k ’”. Scale bar = 20 μm. The histograms show the numbers of EdU + cells g and Myo7a + cells h in the sensory domain and EdU + /Sox2 + cells in the GER (l) in the different groups. The cells were counted per 100 μm length along the cochlea, which was measured between the outer hair cells and the inner hair cells (* p < 0.05, *** p < 0.001), or were counted per 30 μm 2 area of the GER of the cochlea (*** p < 0.001)

Article Snippet: Cultures were exposed to culture medium with 10% FBS for about 2 h and then given the following treatments: DAPT (γ-secretase inhibitor IX, N -[ N -(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butylester, EMD, Gibbstown, NJ, USA); QS11 ((2S)-2-[2-(indan-5-yloxy)-9-(1,1′-biphenyl-4-yl) methyl]-9H–purin-6-ylamino)-3-phenyl-propan-1-ol, Tocris Biosciences, Ellisville, Missouri, USA), which modulates ARF-GTP levels and synergizes with the Wnt/β-catenin signaling pathway to upregulate β-catenin nuclear translocation; and recombinant SHH protein (R & D Systems, Minneapolis, MN, USA), which activates the SHH signaling pathway.

Techniques: In Vitro

Journal: Cell and Tissue Research

Article Title: The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea

doi: 10.1007/s00441-021-03493-w

Figure Lengend Snippet: The role of SHH with DAPT and QS11 in the damaged inner ear in vitro. Neonatal P0–P1 cochleae were cultured with 1.0 mM neomycin for 16 h and then were treated with either 200 ng/ml SHH a – a ”, d – d ”, 5 μM DAPT, or 10 μM QS11 b – b ”, e – e ” or the combination of all three c – c ”, f – f ” for 7 days. There were some EdU + /Sox2 + cells and EdU + /Myo7a + cells in the sensory area of the cochlea. The numbers of EdU + cells in the DAPT + QS11 + SHH group were greater than those in the DAPT + QS11 group a – a ”, b – b ”, c – c ”, especially in the apical turn of the cochlea d – d ”, e – e ”, f – f ” (scale bar = 20 μm). The histograms show the numbers of Myo7a + cells d and EdU + cells e in these groups. The cells were counted per 100-μm length along the cochlea, which was measured between the outer hair cells and the inner hair cells (* p < 0.05, *** p < 0.001)

Article Snippet: Cultures were exposed to culture medium with 10% FBS for about 2 h and then given the following treatments: DAPT (γ-secretase inhibitor IX, N -[ N -(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butylester, EMD, Gibbstown, NJ, USA); QS11 ((2S)-2-[2-(indan-5-yloxy)-9-(1,1′-biphenyl-4-yl) methyl]-9H–purin-6-ylamino)-3-phenyl-propan-1-ol, Tocris Biosciences, Ellisville, Missouri, USA), which modulates ARF-GTP levels and synergizes with the Wnt/β-catenin signaling pathway to upregulate β-catenin nuclear translocation; and recombinant SHH protein (R & D Systems, Minneapolis, MN, USA), which activates the SHH signaling pathway.

Techniques: In Vitro, Cell Culture

Journal: Cell and Tissue Research

Article Title: The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea

doi: 10.1007/s00441-021-03493-w

Figure Lengend Snippet: Gene expression and related mechanisms involved in SC proliferation and HC regeneration. DE genes in the neomycin control, Neo + SHH, Neo + DAPT + QS11, and Neo + DAPT + QS11 + SHH groups a . Red represents upregulated expression levels and blue represents downregulated expression levels. Each row represents one gene, and each column represents one experimental group. The heatmap shows the expression of DE genes involved in the cell cycle b , TGF-β c , Hippo d , transcription factor e , Notch f , Wnt g , and SHH h signaling pathways

Article Snippet: Cultures were exposed to culture medium with 10% FBS for about 2 h and then given the following treatments: DAPT (γ-secretase inhibitor IX, N -[ N -(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butylester, EMD, Gibbstown, NJ, USA); QS11 ((2S)-2-[2-(indan-5-yloxy)-9-(1,1′-biphenyl-4-yl) methyl]-9H–purin-6-ylamino)-3-phenyl-propan-1-ol, Tocris Biosciences, Ellisville, Missouri, USA), which modulates ARF-GTP levels and synergizes with the Wnt/β-catenin signaling pathway to upregulate β-catenin nuclear translocation; and recombinant SHH protein (R & D Systems, Minneapolis, MN, USA), which activates the SHH signaling pathway.

Techniques: Gene Expression, Control, Expressing, Protein-Protein interactions

Journal: Cell and Tissue Research

Article Title: The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea

doi: 10.1007/s00441-021-03493-w

Figure Lengend Snippet: Differential expression analysis between groups. Gene ontology analysis was performed with the DE genes between the Neo + DAPT + QS11 and Neo + DAPT + QS11 + SHH groups a . The results of qRT-PCR of some of the DE genes showed differential expression in the Neomycin control, Neo + SHH, Neo + DAPT + QS11, and Neo + DAPT + QS11 + SHH groups, and these differences were consistent with the RNA-Seq results b

Article Snippet: Cultures were exposed to culture medium with 10% FBS for about 2 h and then given the following treatments: DAPT (γ-secretase inhibitor IX, N -[ N -(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butylester, EMD, Gibbstown, NJ, USA); QS11 ((2S)-2-[2-(indan-5-yloxy)-9-(1,1′-biphenyl-4-yl) methyl]-9H–purin-6-ylamino)-3-phenyl-propan-1-ol, Tocris Biosciences, Ellisville, Missouri, USA), which modulates ARF-GTP levels and synergizes with the Wnt/β-catenin signaling pathway to upregulate β-catenin nuclear translocation; and recombinant SHH protein (R & D Systems, Minneapolis, MN, USA), which activates the SHH signaling pathway.

Techniques: Quantitative Proteomics, Quantitative RT-PCR, Control, RNA Sequencing

Journal: Nature Communications

Article Title: An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism

doi: 10.1038/s41467-021-21847-4

Figure Lengend Snippet: a Western blot of cells expressing Scr, ARF5 or ARF6 shRNA using anti-ARF5, ARF6 and GAPDH (loading control) antibodies. ARF intensity normalised to Scr quantified. Mean ± s.d., n = 3 independent experiments. p -values, one-way ANOVA. ** p ≤ 0.01. b and c Phase images of acini (mVenus positive, yellow) described in ( a ). Scale bars, 100 μm. Heatmap, area and compactness measurements Z -score-normalised to control. p -values; one-way ANOVA, greyscale values as indicated. n = 3 independent experiments, 5 replicates/condition, 2880–3188 acini/condition in total. Cartoon, acini phenotype representative of each condition. d Western blot of cells co-overexpressing (OX) mNeonGreen (mNG) and TagRFP-T (RFP) or ARF5-mNG and ARF6-RFP (ARF5/6), and Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, ARF5, ARF6 and GAPDH (loading control for ARF5) antibodies used. n = 2 independent experiments. e and f Phase images of acini described in ( d ). Scale bars, 100 μm. Heatmap, area and compactness measurements Z -score-normalised to control. p -values, one-way ANOVA, greyscale values as indicated. n = 2 independent experiments, 4 replicates/condition, 1254–1567 acini/condition in total. g Cells co-overexpressing ARF5-mNG and ARF6-RFP (ARF5/6 OX) were stained for IQSEC1 v2. Merged and magnified image of spindle cell shown. Arrowheads, colocalization. n = 3 independent experiments. Scale bars, 20 μm. h Acini expressing ARF5-mNG or ARF6-mNG were stained for IQSEC1 (FIRE LUT). Yellow and white arrowheads, colocalization in juxtanuclear region and protrusive tips, respectively. Arrows, lack of colocalization. n = 3 independent acini imaged. Scale bars, 5 μm. i Schema, GTPase cycle, site of action of NAV-2729, QS11 and GTP-loaded ARF detection by GGA1-NGAT. j Acini expressing ARF5-mNG or ARF6-mNG and GGA1-NGAT-RFP were fixed and FIRE LUT of maximum projections and a single Z-slice is shown. Arrowheads, colocalization in protrusions, arrows, colocalization in cell body. n = 2–5 independent acini imaged. Scale bars, 10 μm. k and l Cells expressing GGA1-NGAT and k ARF5-NG or l ARF6-NG were transfected with Scr or IQSEC1 KD4 shRNA then treated with NAV-2729 or QS11. % overlap of ARF and ARF-GTP probe/cell is shown in box-and-whiskers plot: 10–90 percentile; + mean; dots, outliers; midline, median; boundaries, quartiles. n = 2 independent experiments, 3 replicates/condition with k 541–1287 and l 390–798 cells quantified/condition. p -values, one-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. m Schema, localisation of IQSEC1 and active ARFs in protrusions.

Article Snippet: Growth factors or inhibitors were added as follows; 50 ng/ml hepatocyte growth factor (HGF) (PeproTech), 10 μM NAV-2729 (Glixx Laboratories), 10 μM QS11 (Tocris), 100 μM chloroquine (CST), 20 μM SecinH3 (Tocris), 25 μM cycloheximide (Sigma), 1 μM LY-294002 (Merck), 1 μM AZD8835 (AstraZeneca), 0.1 μM AZD8186 (AstraZeneca), 1 μM AS-6052-40 (Stratech), 1 μM Cal-101 (Stratech), and 10 μM AktII (Calbiochem) inhibitors.

Techniques: Western Blot, Expressing, shRNA, Control, Staining, Transfection