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culture atcc 90494  (ATCC)


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    Structured Review

    ATCC culture atcc 90494
    Culture Atcc 90494, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
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    ATCC culture atcc 90494
    Culture Atcc 90494, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology pcdh15 shrna b
    a – c Transverse spinal cord sections from embryonic day 16.5 (E16.5) mice were used to perform in situ hybridization to detect Pdgfrα or <t>Pcdh15</t> mRNA. a E16.5 mouse spinal cord shows no labelling in the absence of an RNA probe. Line denotes the border of the spinal cord grey and white matter. b Pdgfrα + OPCs are readily detected in the E16.5 spinal cord white matter. The dashed box denotes the white matter region enlarged in the lower right corner. c Image showing Pcdh15 + cells in the E16.5 mouse spinal cord. The dashed box denotes the white matter region enlarged in the lower right corner. d Quantification of Pcdh15 mRNA (in FPKM, Fragments Per Kilobase Million) in neurons and glial cells acutely purified from the early postnatal mouse brain. Graph generated using previously published and publicly available RNA sequencing data from Zhang et al. . e Genome browser tracks of the Pcdh15 locus showing RNA-Seq alignments from nuclear RNA purified from the brains of adult Ng2-CreER T2 :: Sun1-eGFP or PLP-CreERT:: Sun1-eGFP mice. Pcdh15 mRNA is highly expressed by Ng2 + OPCs and most transcripts correspond to the CD3 isoforms of Pcdh15. f – h Immunocytochemistry to detect GFP (blue), PDGFRα (green) and Pcdh15 (red) in primary OPCs generated from the early postnatal Pdgfrα-histGFP mouse cortex. Scale bars represent 60 µm ( a – c ) and 20 µm ( f – h ).
    Pcdh15 Shrna B, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology pcdh15 shrna a
    a – c Transverse spinal cord sections from embryonic day 16.5 (E16.5) mice were used to perform in situ hybridization to detect Pdgfrα or <t>Pcdh15</t> mRNA. a E16.5 mouse spinal cord shows no labelling in the absence of an RNA probe. Line denotes the border of the spinal cord grey and white matter. b Pdgfrα + OPCs are readily detected in the E16.5 spinal cord white matter. The dashed box denotes the white matter region enlarged in the lower right corner. c Image showing Pcdh15 + cells in the E16.5 mouse spinal cord. The dashed box denotes the white matter region enlarged in the lower right corner. d Quantification of Pcdh15 mRNA (in FPKM, Fragments Per Kilobase Million) in neurons and glial cells acutely purified from the early postnatal mouse brain. Graph generated using previously published and publicly available RNA sequencing data from Zhang et al. . e Genome browser tracks of the Pcdh15 locus showing RNA-Seq alignments from nuclear RNA purified from the brains of adult Ng2-CreER T2 :: Sun1-eGFP or PLP-CreERT:: Sun1-eGFP mice. Pcdh15 mRNA is highly expressed by Ng2 + OPCs and most transcripts correspond to the CD3 isoforms of Pcdh15. f – h Immunocytochemistry to detect GFP (blue), PDGFRα (green) and Pcdh15 (red) in primary OPCs generated from the early postnatal Pdgfrα-histGFP mouse cortex. Scale bars represent 60 µm ( a – c ) and 20 µm ( f – h ).
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    Thermo Fisher aban gibot san 90494
    a – c Transverse spinal cord sections from embryonic day 16.5 (E16.5) mice were used to perform in situ hybridization to detect Pdgfrα or <t>Pcdh15</t> mRNA. a E16.5 mouse spinal cord shows no labelling in the absence of an RNA probe. Line denotes the border of the spinal cord grey and white matter. b Pdgfrα + OPCs are readily detected in the E16.5 spinal cord white matter. The dashed box denotes the white matter region enlarged in the lower right corner. c Image showing Pcdh15 + cells in the E16.5 mouse spinal cord. The dashed box denotes the white matter region enlarged in the lower right corner. d Quantification of Pcdh15 mRNA (in FPKM, Fragments Per Kilobase Million) in neurons and glial cells acutely purified from the early postnatal mouse brain. Graph generated using previously published and publicly available RNA sequencing data from Zhang et al. . e Genome browser tracks of the Pcdh15 locus showing RNA-Seq alignments from nuclear RNA purified from the brains of adult Ng2-CreER T2 :: Sun1-eGFP or PLP-CreERT:: Sun1-eGFP mice. Pcdh15 mRNA is highly expressed by Ng2 + OPCs and most transcripts correspond to the CD3 isoforms of Pcdh15. f – h Immunocytochemistry to detect GFP (blue), PDGFRα (green) and Pcdh15 (red) in primary OPCs generated from the early postnatal Pdgfrα-histGFP mouse cortex. Scale bars represent 60 µm ( a – c ) and 20 µm ( f – h ).
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    a – c Transverse spinal cord sections from embryonic day 16.5 (E16.5) mice were used to perform in situ hybridization to detect Pdgfrα or Pcdh15 mRNA. a E16.5 mouse spinal cord shows no labelling in the absence of an RNA probe. Line denotes the border of the spinal cord grey and white matter. b Pdgfrα + OPCs are readily detected in the E16.5 spinal cord white matter. The dashed box denotes the white matter region enlarged in the lower right corner. c Image showing Pcdh15 + cells in the E16.5 mouse spinal cord. The dashed box denotes the white matter region enlarged in the lower right corner. d Quantification of Pcdh15 mRNA (in FPKM, Fragments Per Kilobase Million) in neurons and glial cells acutely purified from the early postnatal mouse brain. Graph generated using previously published and publicly available RNA sequencing data from Zhang et al. . e Genome browser tracks of the Pcdh15 locus showing RNA-Seq alignments from nuclear RNA purified from the brains of adult Ng2-CreER T2 :: Sun1-eGFP or PLP-CreERT:: Sun1-eGFP mice. Pcdh15 mRNA is highly expressed by Ng2 + OPCs and most transcripts correspond to the CD3 isoforms of Pcdh15. f – h Immunocytochemistry to detect GFP (blue), PDGFRα (green) and Pcdh15 (red) in primary OPCs generated from the early postnatal Pdgfrα-histGFP mouse cortex. Scale bars represent 60 µm ( a – c ) and 20 µm ( f – h ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a – c Transverse spinal cord sections from embryonic day 16.5 (E16.5) mice were used to perform in situ hybridization to detect Pdgfrα or Pcdh15 mRNA. a E16.5 mouse spinal cord shows no labelling in the absence of an RNA probe. Line denotes the border of the spinal cord grey and white matter. b Pdgfrα + OPCs are readily detected in the E16.5 spinal cord white matter. The dashed box denotes the white matter region enlarged in the lower right corner. c Image showing Pcdh15 + cells in the E16.5 mouse spinal cord. The dashed box denotes the white matter region enlarged in the lower right corner. d Quantification of Pcdh15 mRNA (in FPKM, Fragments Per Kilobase Million) in neurons and glial cells acutely purified from the early postnatal mouse brain. Graph generated using previously published and publicly available RNA sequencing data from Zhang et al. . e Genome browser tracks of the Pcdh15 locus showing RNA-Seq alignments from nuclear RNA purified from the brains of adult Ng2-CreER T2 :: Sun1-eGFP or PLP-CreERT:: Sun1-eGFP mice. Pcdh15 mRNA is highly expressed by Ng2 + OPCs and most transcripts correspond to the CD3 isoforms of Pcdh15. f – h Immunocytochemistry to detect GFP (blue), PDGFRα (green) and Pcdh15 (red) in primary OPCs generated from the early postnatal Pdgfrα-histGFP mouse cortex. Scale bars represent 60 µm ( a – c ) and 20 µm ( f – h ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: In Situ Hybridization, Purification, Generated, RNA Sequencing Assay, Immunocytochemistry

    a Western blot gel image showing Pcdh15 expression in protein lysates generated from adult mouse brain ( n = 3 mice). b Western blot gel image showing control- and Pcdh15-shRNA treated 9DIV OPC cultures, 12 h after transfection. c Expression (pixel integrated density) of the ~62 KDa Pcdh15 variant was quantified relative to the expression of β-actin in protein lysates from control and Pcdh15 knockdown OPCs, 12 h after transfection. Data are normalised to the relative expression of Pcdh15 in control OPCs for each culture [mean ± SEM, n = 4 independent cultures; one sample t -test for Pcdh15-shRNA: deviation from 100 (%) = −96.02, SD of deviation = 2.54, t (3) = 75.33, p < 0.0001]. d – f Confocal images of control- and Pcdh15 -shRNA treated and untransfected OPCs following immunocytochemistry to detect Pcdh15 (red) and GFP (OPCs, green). g Quantification of Pcdh15 protein expression (immunocytochemistry pixel integrated density) in control- and Pcdh15-shRNA treated and untransfected OPCs [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (2,6) = 33.83, p = 0.0005; Tukey’s multiple comparisons test: *** p = 0.001, control shRNA vs. no transfection p = 0.99]. h – k Confocal images showing OPCs transfected with control shRNA or one of three distinct Pcdh15-shRNAs (Pcdh15-shRNA-A or Pcdh15-shRNA-B or Pcdh15-shRNA-C) for 12 h, before immunocytochemistry was performed to detect Pcdh15 (red) and GFP (OPCs, green). l Quantification of Pcdh15 expression (integrated pixel density) for OPCs transfected with control shRNA, Pcdh15-shRNA-A, Pcdh15-shRNA-B or Pcdh15-shRNA-C [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (3,8) = 31.05, p < 0.0001; Tukey’s multiple comparisons test: *** p < 0.001; Pcdh15-shRNA-A vs. B and B vs. C, p = 0.9; Pcdh15-shRNA-A vs. C p = 0.6]. Scale bars represent 30 µm ( d – f ) or 20 µm ( h – k ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Western blot gel image showing Pcdh15 expression in protein lysates generated from adult mouse brain ( n = 3 mice). b Western blot gel image showing control- and Pcdh15-shRNA treated 9DIV OPC cultures, 12 h after transfection. c Expression (pixel integrated density) of the ~62 KDa Pcdh15 variant was quantified relative to the expression of β-actin in protein lysates from control and Pcdh15 knockdown OPCs, 12 h after transfection. Data are normalised to the relative expression of Pcdh15 in control OPCs for each culture [mean ± SEM, n = 4 independent cultures; one sample t -test for Pcdh15-shRNA: deviation from 100 (%) = −96.02, SD of deviation = 2.54, t (3) = 75.33, p < 0.0001]. d – f Confocal images of control- and Pcdh15 -shRNA treated and untransfected OPCs following immunocytochemistry to detect Pcdh15 (red) and GFP (OPCs, green). g Quantification of Pcdh15 protein expression (immunocytochemistry pixel integrated density) in control- and Pcdh15-shRNA treated and untransfected OPCs [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (2,6) = 33.83, p = 0.0005; Tukey’s multiple comparisons test: *** p = 0.001, control shRNA vs. no transfection p = 0.99]. h – k Confocal images showing OPCs transfected with control shRNA or one of three distinct Pcdh15-shRNAs (Pcdh15-shRNA-A or Pcdh15-shRNA-B or Pcdh15-shRNA-C) for 12 h, before immunocytochemistry was performed to detect Pcdh15 (red) and GFP (OPCs, green). l Quantification of Pcdh15 expression (integrated pixel density) for OPCs transfected with control shRNA, Pcdh15-shRNA-A, Pcdh15-shRNA-B or Pcdh15-shRNA-C [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (3,8) = 31.05, p < 0.0001; Tukey’s multiple comparisons test: *** p < 0.001; Pcdh15-shRNA-A vs. B and B vs. C, p = 0.9; Pcdh15-shRNA-A vs. C p = 0.6]. Scale bars represent 30 µm ( d – f ) or 20 µm ( h – k ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Western Blot, Expressing, Generated, shRNA, Transfection, Variant Assay, Immunocytochemistry

    a – f Confocal images showing control ( a – c ) or Pcdh15 knockdown ( d – f ) GFP + OPCs (green) after 12 h of EdU labelling (red). White arrowheads indicate EdU + GFP + OPCs. g The proportion of control and Pcdh15 knockdown GFP + OPCs (%) that incorporated EdU (EdU + GFP + /GFP + × 100; mean ± SEM, n = 3 independent cultures; unpaired t -test, * p = 0.048). h The proportion (%) of GFP + OPCs that incorporate EdU (EdU + GFP + /GFP + × 100) in cultures transfected with control-shRNA, Pcdh15-shRNA-A, Pcdh15-shRNA-B or Pcdh15-shRNA-C [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (3,8) = 5.658, p = 0.02; Tukey’s multiple comparisons test: Control shRNA vs Pch15 shRNA-A, * p < 0.05; Pcdh15-shRNA-A vs. Pcdh15-shRNA-B, p = 1.0; Pcdh15-shRNA-A vs. Pcdh15-shRNA-C, p = 1.0, Pcdh15-shRNA-B vs. Pcdh15-shRNA-C, p = 0.99]. i – p Confocal images showing EdU-labelling (red) in GFP + OPCs (green) transfected with control-shRNA ( i , j ), Pcdh15-shRNA-A ( k , l ), Pcdh15-shRNA-B ( m , n ) or Pcdh15-shRNA-C ( o , p ). q The proportion (%) of EdU + GFP + nuclei that belonged to: single cells (nuclei not contacting others); paired cells (two nuclei in contact), or clustered cells (groups of three or four nuclei in contact with each other) [mean ± SEM for n = 3 independent cultures; ≥86 EdU + GFP + cells analysed per coverslip. Two-way ANOVA: shRNA F (1, 16) = 0.0037, p = 0.9524; cluster F (3, 16) = 6206, p < 0.0001; interaction F (3, 16) = 35.79, p < 0.0001; with Bonferroni multiple comparison test: **** p < 0.0001. r Quantification of the mean Euclidean distance between EdU + GFP + nuclei (mean ± SEM, n = 3 independent cultures; ≥86 EdU + GFP + cells analysed per coverslip, unpaired t -test with welch’s correction, * p = 0.024). Scale bars represent 25 µm ( a – f ) or 15 µm ( i – l ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a – f Confocal images showing control ( a – c ) or Pcdh15 knockdown ( d – f ) GFP + OPCs (green) after 12 h of EdU labelling (red). White arrowheads indicate EdU + GFP + OPCs. g The proportion of control and Pcdh15 knockdown GFP + OPCs (%) that incorporated EdU (EdU + GFP + /GFP + × 100; mean ± SEM, n = 3 independent cultures; unpaired t -test, * p = 0.048). h The proportion (%) of GFP + OPCs that incorporate EdU (EdU + GFP + /GFP + × 100) in cultures transfected with control-shRNA, Pcdh15-shRNA-A, Pcdh15-shRNA-B or Pcdh15-shRNA-C [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (3,8) = 5.658, p = 0.02; Tukey’s multiple comparisons test: Control shRNA vs Pch15 shRNA-A, * p < 0.05; Pcdh15-shRNA-A vs. Pcdh15-shRNA-B, p = 1.0; Pcdh15-shRNA-A vs. Pcdh15-shRNA-C, p = 1.0, Pcdh15-shRNA-B vs. Pcdh15-shRNA-C, p = 0.99]. i – p Confocal images showing EdU-labelling (red) in GFP + OPCs (green) transfected with control-shRNA ( i , j ), Pcdh15-shRNA-A ( k , l ), Pcdh15-shRNA-B ( m , n ) or Pcdh15-shRNA-C ( o , p ). q The proportion (%) of EdU + GFP + nuclei that belonged to: single cells (nuclei not contacting others); paired cells (two nuclei in contact), or clustered cells (groups of three or four nuclei in contact with each other) [mean ± SEM for n = 3 independent cultures; ≥86 EdU + GFP + cells analysed per coverslip. Two-way ANOVA: shRNA F (1, 16) = 0.0037, p = 0.9524; cluster F (3, 16) = 6206, p < 0.0001; interaction F (3, 16) = 35.79, p < 0.0001; with Bonferroni multiple comparison test: **** p < 0.0001. r Quantification of the mean Euclidean distance between EdU + GFP + nuclei (mean ± SEM, n = 3 independent cultures; ≥86 EdU + GFP + cells analysed per coverslip, unpaired t -test with welch’s correction, * p = 0.024). Scale bars represent 25 µm ( a – f ) or 15 µm ( i – l ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Transfection, shRNA

    a Primary mouse OPC cultures were transfected with control- or Pcdh15-shRNA and protein lysates collected 12 h later, at 10 DIV. Image of a Western blot performed to detect phosphorylated β-catenin (p-β-catenin), active unphosphorylated β-catenin, and β-actin. b Quantification of p-β-catenin (Ser33/Ser37/Thr41) expression by OPC cultures transfected with control- or Pcdh15-shRNA, normalised to β-actin expression (mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.17). c Quantification of β-catenin expression by OPC cultures, 12 h after transfection with control- or Pcdh15-shRNA, normalised to β-actin expression (mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.63). d Western blot gel image showing ERK1/2, phosphorylated ERK1/2, β-actin, and GAPDH expression by OPCs transfected with control- or Pcdh15-shRNA. e Western blot protein band integrated pixel density was quantified for p-ERK1/2 and ERK1/2 and used to calculate the proportion of ERK1 that was phosphorylated (p-ERK1/ERK1) and the proportion of ERK2 that was phosphorylated (p-ERK2/ERK2) for OPCs transfected with control- or Pcdh15-shRNA [mean ± SEM, n = 3 independent cultures; p-ERK1/ERK1 and p-ERK2/ERK2 data were analysed separately by unpaired t -test (note // break to x -axis), * p < 0.05, ** p < 0.01]. f Western blot gel image showing ERK1/2, p-ERK1/2, β-actin, and GAPDH expression by OPCs transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126 (10 µM). g Quantification of the proportion of ERK1 that was phosphorylated (integrated pixel density for p-ERK1/integrated pixel density for ERK1) and the proportion of ERK2 that was phosphorylated (integrated pixel density p-ERK2/integrated pixel density for ERK2) in OPCs transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126. For each culture, these data were normalised to the control shRNA group, i.e., relative protein level/control shRNA relative protein level × 100 [mean ± SEM, n = 3 independent cultures; two‐way ANOVA for p-ERK1/ERK1: shRNA treatment F (1,8) = 11.06, p = 0.0104; drug treatment F (1,8) = 107.9, p < 0.0001; interaction F (1,8) = 10.18, p = 0.012 with Bonferroni multiple comparisons, ** p < 0.01, **** p < 0.0001; two-way ANOVA for p-ERK2/ERK2: shRNA treatment F (1,8) = 2.12, p = 0.061; drug treatment F (1,8) = 91.19, p < 0.0001; interaction F (1,8) = 3.03, p = 0.032 with Bonferroni multiple comparisons, * p < 0.05, **** p < 0.0001]. h – k Confocal images showing GFP + OPCs (green) transfected with control- or Pcdh15 -shRNA, treated with DMSO or U0126 and exposed to EdU (red) for 12 h. l The proportion (%) of GFP + control or Pcdh15 knockdown OPCs that incorporate EdU following treatment with DMSO or U0126 [mean ± SEM, n = 3 independent cultures; two‐way ANOVA: shRNA F (1,8) = 13.0, p = 0.007; drug treatment F (1,8) = 62.98, p < 0.0001; interaction F (1,8) = 6.91, p = 0.03 with Bonferroni multiple comparisons test: * p < 0.05, *** p < 0.001]. Scale bars represent 35 µm.

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Primary mouse OPC cultures were transfected with control- or Pcdh15-shRNA and protein lysates collected 12 h later, at 10 DIV. Image of a Western blot performed to detect phosphorylated β-catenin (p-β-catenin), active unphosphorylated β-catenin, and β-actin. b Quantification of p-β-catenin (Ser33/Ser37/Thr41) expression by OPC cultures transfected with control- or Pcdh15-shRNA, normalised to β-actin expression (mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.17). c Quantification of β-catenin expression by OPC cultures, 12 h after transfection with control- or Pcdh15-shRNA, normalised to β-actin expression (mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.63). d Western blot gel image showing ERK1/2, phosphorylated ERK1/2, β-actin, and GAPDH expression by OPCs transfected with control- or Pcdh15-shRNA. e Western blot protein band integrated pixel density was quantified for p-ERK1/2 and ERK1/2 and used to calculate the proportion of ERK1 that was phosphorylated (p-ERK1/ERK1) and the proportion of ERK2 that was phosphorylated (p-ERK2/ERK2) for OPCs transfected with control- or Pcdh15-shRNA [mean ± SEM, n = 3 independent cultures; p-ERK1/ERK1 and p-ERK2/ERK2 data were analysed separately by unpaired t -test (note // break to x -axis), * p < 0.05, ** p < 0.01]. f Western blot gel image showing ERK1/2, p-ERK1/2, β-actin, and GAPDH expression by OPCs transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126 (10 µM). g Quantification of the proportion of ERK1 that was phosphorylated (integrated pixel density for p-ERK1/integrated pixel density for ERK1) and the proportion of ERK2 that was phosphorylated (integrated pixel density p-ERK2/integrated pixel density for ERK2) in OPCs transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126. For each culture, these data were normalised to the control shRNA group, i.e., relative protein level/control shRNA relative protein level × 100 [mean ± SEM, n = 3 independent cultures; two‐way ANOVA for p-ERK1/ERK1: shRNA treatment F (1,8) = 11.06, p = 0.0104; drug treatment F (1,8) = 107.9, p < 0.0001; interaction F (1,8) = 10.18, p = 0.012 with Bonferroni multiple comparisons, ** p < 0.01, **** p < 0.0001; two-way ANOVA for p-ERK2/ERK2: shRNA treatment F (1,8) = 2.12, p = 0.061; drug treatment F (1,8) = 91.19, p < 0.0001; interaction F (1,8) = 3.03, p = 0.032 with Bonferroni multiple comparisons, * p < 0.05, **** p < 0.0001]. h – k Confocal images showing GFP + OPCs (green) transfected with control- or Pcdh15 -shRNA, treated with DMSO or U0126 and exposed to EdU (red) for 12 h. l The proportion (%) of GFP + control or Pcdh15 knockdown OPCs that incorporate EdU following treatment with DMSO or U0126 [mean ± SEM, n = 3 independent cultures; two‐way ANOVA: shRNA F (1,8) = 13.0, p = 0.007; drug treatment F (1,8) = 62.98, p < 0.0001; interaction F (1,8) = 6.91, p = 0.03 with Bonferroni multiple comparisons test: * p < 0.05, *** p < 0.001]. Scale bars represent 35 µm.

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Transfection, shRNA, Western Blot, Expressing

    12 h after OPCs were transfected with control or Pcdh15 -shRNA, they were imaged once a minute for 2 h and the resulting time-lapse videos were used to quantify different aspects of OPC motility. a Image series show a filopodial contact (black arrowhead) between adjacent OPCs in a control and Pcdh15 knockdown culture. All images are time-stamped relative to the first image in the depicted series (designated as 0 min). b The mean duration (min) that a filopodial contact was sustained in control and Pcdh15 knockdown cultures (mean ± SEM, n = 3 independent cultures, ≥66 filopodial contacts measured per condition; unpaired t test, * p = 0.034). c Excepts from a time-lapse image series showing a control OPC process as it extrudes and retracts a veil and a Pcdh15 knockdown OPC as it extrudes and maintains its veil. The images are time-stamped relative to the first image in the depicted series (designated as 0 min). Black arrowheads indicate elaborated veils. d Quantification of the mean veiling time for OPC processes in control- and Pcdh15 -shRNA treated cultures (time taken to complete an extrusion and retraction) (mean ± SEM, n = 3 independent cultures, ≥146 veils analysed per condition; unpaired t -test, ** p = 0.004). e Images of control- and Pcdh15 -shRNA treated OPCs, 12 h after transfection. f Quantification of the mean number of processes supported by control and Pcdh15 shRNA-treated OPCs (mean ± SEM, n = 3 independent cultures and ≥81 OPCs analysed per condition; unpaired t test, *** p = 0.0004). g Short image series extracted from the time-lapse videos to show control and Pcdh15 knockdown OPCs extending a new process. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and the white asterisks denote the new processes. h Quantification of the mean number of new processes elaborated by control- and Pcdh15 -shRNA transfected OPCs each hour (mean ± SEM, n = 3 independent cultures and ≥35 OPCs analysed per condition; unpaired t test, * p = 0.048). i The migration trajectories for the soma of 52 control- or Pcdh15 -shRNA treated OPCs. j Mean migration speed for the soma of control- and Pcdh15 -shRNA treated OPCs (mean ± SEM, n = 52 OPCs per group, unpaired t tests with Welch’s correction, **** p < 0.0001). k Mean distance that each OPC soma moved (path length) in control- and Pcdh15 -shRNA treated cultures (mean ± SEM, n = 52 OPCs per group, unpaired t tests with Welch’s correction, **** p < 0.0001). l Mean Euclidean distance (shortest distance between the start and end points) that each OPC soma moved in control- or Pcdh15 -shRNA treated cultures (mean ± SEM, unpaired t tests with Welch’s correction, *** p < 0.0002). Scale bars represent 5 µm ( a , c ), 8 µm ( g ) or 20 µm ( e ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: 12 h after OPCs were transfected with control or Pcdh15 -shRNA, they were imaged once a minute for 2 h and the resulting time-lapse videos were used to quantify different aspects of OPC motility. a Image series show a filopodial contact (black arrowhead) between adjacent OPCs in a control and Pcdh15 knockdown culture. All images are time-stamped relative to the first image in the depicted series (designated as 0 min). b The mean duration (min) that a filopodial contact was sustained in control and Pcdh15 knockdown cultures (mean ± SEM, n = 3 independent cultures, ≥66 filopodial contacts measured per condition; unpaired t test, * p = 0.034). c Excepts from a time-lapse image series showing a control OPC process as it extrudes and retracts a veil and a Pcdh15 knockdown OPC as it extrudes and maintains its veil. The images are time-stamped relative to the first image in the depicted series (designated as 0 min). Black arrowheads indicate elaborated veils. d Quantification of the mean veiling time for OPC processes in control- and Pcdh15 -shRNA treated cultures (time taken to complete an extrusion and retraction) (mean ± SEM, n = 3 independent cultures, ≥146 veils analysed per condition; unpaired t -test, ** p = 0.004). e Images of control- and Pcdh15 -shRNA treated OPCs, 12 h after transfection. f Quantification of the mean number of processes supported by control and Pcdh15 shRNA-treated OPCs (mean ± SEM, n = 3 independent cultures and ≥81 OPCs analysed per condition; unpaired t test, *** p = 0.0004). g Short image series extracted from the time-lapse videos to show control and Pcdh15 knockdown OPCs extending a new process. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and the white asterisks denote the new processes. h Quantification of the mean number of new processes elaborated by control- and Pcdh15 -shRNA transfected OPCs each hour (mean ± SEM, n = 3 independent cultures and ≥35 OPCs analysed per condition; unpaired t test, * p = 0.048). i The migration trajectories for the soma of 52 control- or Pcdh15 -shRNA treated OPCs. j Mean migration speed for the soma of control- and Pcdh15 -shRNA treated OPCs (mean ± SEM, n = 52 OPCs per group, unpaired t tests with Welch’s correction, **** p < 0.0001). k Mean distance that each OPC soma moved (path length) in control- and Pcdh15 -shRNA treated cultures (mean ± SEM, n = 52 OPCs per group, unpaired t tests with Welch’s correction, **** p < 0.0001). l Mean Euclidean distance (shortest distance between the start and end points) that each OPC soma moved in control- or Pcdh15 -shRNA treated cultures (mean ± SEM, unpaired t tests with Welch’s correction, *** p < 0.0002). Scale bars represent 5 µm ( a , c ), 8 µm ( g ) or 20 µm ( e ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Transfection, shRNA, Migration

    a Image series extracted from time-lapse videos showing filopodial contacts (arrows) formed between adjacent OPCs in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126 (10 µM). All images are time-stamped relative to the first image in the depicted series (designated as 0 min). b The mean duration (min) that filopodial contacts were sustained once they formed between adjacent OPCs in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥44 filopodial contacts measured per condition; two‐way ANOVA: shRNA treatment F (1,8) = 56.29, p < 0.0001; drug treatment F (1,8) = 0.0015, p = 0.96; interaction F (1,8) = 0.00017, p = 0.98 with Bonferroni multiple comparisons test: ** p < 0.01]. c Mean OPC process veiling time (min), i.e., the time taken for an OPC process to complete one full extrusion and retraction of its veil in control- or Pcdh15 -shRNA cultures treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥102 veils analysed per condition; two‐way ANOVA: shRNA treatment F (1,8) = 52.44, p < 0.0001; drug treatment F (1,8) = 0.36, p = 0.56; interaction F (1,8) = 1.05, p = 0.33 with Bonferroni multiple comparisons, ** p < 0.01, *** p < 0.001]. d Image series extracted from time-lapse videos showing OPC processes elaborating and retracting veils in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126. White arrows indicate extruded veils. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight veil extrusion and retraction. e Images of OPCs 12 h after transfection with control- or Pcdh15-shRNA and treatment with DMSO or U0126 (10 µM). f Quantification of the number of new processes elaborated by OPCs each hour in cultures transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥31 OPCs analysed per condition; two‐way ANOVA: shRNA treatment F (1,8) = 314.7, p < 0.0001; drug treatment F (1,8) = 0.48, p = 0.50; interaction F (1,8) = 0.0007, p = 0.97 with Bonferroni multiple comparisons, **** p < 0.0001]. Image series depicting OPC process generation can be found in Supplementary Fig. . Scale bars represent 5 µm ( a , d ) or 10 µm ( e ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Image series extracted from time-lapse videos showing filopodial contacts (arrows) formed between adjacent OPCs in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126 (10 µM). All images are time-stamped relative to the first image in the depicted series (designated as 0 min). b The mean duration (min) that filopodial contacts were sustained once they formed between adjacent OPCs in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥44 filopodial contacts measured per condition; two‐way ANOVA: shRNA treatment F (1,8) = 56.29, p < 0.0001; drug treatment F (1,8) = 0.0015, p = 0.96; interaction F (1,8) = 0.00017, p = 0.98 with Bonferroni multiple comparisons test: ** p < 0.01]. c Mean OPC process veiling time (min), i.e., the time taken for an OPC process to complete one full extrusion and retraction of its veil in control- or Pcdh15 -shRNA cultures treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥102 veils analysed per condition; two‐way ANOVA: shRNA treatment F (1,8) = 52.44, p < 0.0001; drug treatment F (1,8) = 0.36, p = 0.56; interaction F (1,8) = 1.05, p = 0.33 with Bonferroni multiple comparisons, ** p < 0.01, *** p < 0.001]. d Image series extracted from time-lapse videos showing OPC processes elaborating and retracting veils in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126. White arrows indicate extruded veils. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight veil extrusion and retraction. e Images of OPCs 12 h after transfection with control- or Pcdh15-shRNA and treatment with DMSO or U0126 (10 µM). f Quantification of the number of new processes elaborated by OPCs each hour in cultures transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥31 OPCs analysed per condition; two‐way ANOVA: shRNA treatment F (1,8) = 314.7, p < 0.0001; drug treatment F (1,8) = 0.48, p = 0.50; interaction F (1,8) = 0.0007, p = 0.97 with Bonferroni multiple comparisons, **** p < 0.0001]. Image series depicting OPC process generation can be found in Supplementary Fig. . Scale bars represent 5 µm ( a , d ) or 10 µm ( e ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: shRNA, Transfection

    a Confocal images of OPC processes in control- and Pcdh15 shRNA treated cultures immunolabelled to detect microtubules (α-tubulin, green) and F-actin (Phalloidin, red). b Quantification of phalloidin intensity (integrated pixel density) in control and Pcdh15-knockdown OPCs (normalized to the average intensity of control OPC cultures) ( n = 3 independent cultures, ≥51 veils analysed per condition; one-sample t -test for Pcdh15-shRNA: deviation from 100(%) = 30.25, SD of deviation = 9.19, t (2) = 5.697, p = 0.029). c Degree of colocalization between actin (phalloidin) and microtubule (α-actin) labelling in control and Pcdh15 knockdown OPC veils. Colocalization is expressed as a Pearson correlation coefficient (mean ± SEM, n = 3 independent cultures, ≥37 veils measured per condition; unpaired t -test, p = 0.81). d Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect Akt, phosphorylated Akt (p-Akt) and β-actin. e Expression of Akt in control and Pcdh15 knockdown OPC cultures, normalised to β-actin expression (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.12). f Quantification of p-Akt (Ser473) and Akt in control and Pcdh15 knockdown OPCs, normalised to β-actin (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.20). g Average fold change in cytoskeletal gene expression between OPCs transfected with control- and Pcdh15 -shRNA (average from n = 3 independent cultures). h Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect CrkII and WAVE2, which are upstream of the Arp2/3 complex, Arp2, Arp3 and Arpc3, which are components of the Arp2/3 complex, and GAPDH. i CrkII, j WAVE2, k Arp2, l Arp3, and m Arpc3 protein expression was quantified by measuring the integrated pixel density of the corresponding Western blot band and normalising expression to GAPDH expression for each sample [mean ± SEM, n = 3 independent cultures, expression in control and Pcdh15 knockdown OPCs was compared by unpaired t -tests: i p = 0.039, j p = 0.004, k p = 0.0001, l p = 0.028, m p = 0.016]. Scale bars represent 5 µm.

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Confocal images of OPC processes in control- and Pcdh15 shRNA treated cultures immunolabelled to detect microtubules (α-tubulin, green) and F-actin (Phalloidin, red). b Quantification of phalloidin intensity (integrated pixel density) in control and Pcdh15-knockdown OPCs (normalized to the average intensity of control OPC cultures) ( n = 3 independent cultures, ≥51 veils analysed per condition; one-sample t -test for Pcdh15-shRNA: deviation from 100(%) = 30.25, SD of deviation = 9.19, t (2) = 5.697, p = 0.029). c Degree of colocalization between actin (phalloidin) and microtubule (α-actin) labelling in control and Pcdh15 knockdown OPC veils. Colocalization is expressed as a Pearson correlation coefficient (mean ± SEM, n = 3 independent cultures, ≥37 veils measured per condition; unpaired t -test, p = 0.81). d Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect Akt, phosphorylated Akt (p-Akt) and β-actin. e Expression of Akt in control and Pcdh15 knockdown OPC cultures, normalised to β-actin expression (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.12). f Quantification of p-Akt (Ser473) and Akt in control and Pcdh15 knockdown OPCs, normalised to β-actin (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.20). g Average fold change in cytoskeletal gene expression between OPCs transfected with control- and Pcdh15 -shRNA (average from n = 3 independent cultures). h Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect CrkII and WAVE2, which are upstream of the Arp2/3 complex, Arp2, Arp3 and Arpc3, which are components of the Arp2/3 complex, and GAPDH. i CrkII, j WAVE2, k Arp2, l Arp3, and m Arpc3 protein expression was quantified by measuring the integrated pixel density of the corresponding Western blot band and normalising expression to GAPDH expression for each sample [mean ± SEM, n = 3 independent cultures, expression in control and Pcdh15 knockdown OPCs was compared by unpaired t -tests: i p = 0.039, j p = 0.004, k p = 0.0001, l p = 0.028, m p = 0.016]. Scale bars represent 5 µm.

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: shRNA, Western Blot, Expressing, Transfection

    a – c Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and Hoechst 33342 (HST; blue). d Single confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and Pcdh15 (green). e – g Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and HST (blue). h Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and Pcdh15 (green). i – k Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red) and the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). l Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and the CD3 isoforms of Pcdh15 (green). (m-o) Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). p Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and the CD3 isoforms of Pcdh15 (green). Scale bars represent 20 µm ( a – c , e – g , i – k , m – o ) or 5 µm ( d , h , l , p ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a – c Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and Hoechst 33342 (HST; blue). d Single confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and Pcdh15 (green). e – g Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and HST (blue). h Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and Pcdh15 (green). i – k Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red) and the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). l Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and the CD3 isoforms of Pcdh15 (green). (m-o) Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). p Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and the CD3 isoforms of Pcdh15 (green). Scale bars represent 20 µm ( a – c , e – g , i – k , m – o ) or 5 µm ( d , h , l , p ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Expressing

    a A schematic proposing a mechanism by which Pcdh15 could impact actin polymerisation in OPCs and depicting the point of action in the pathway of the drugs ML141 and CK666. b – m Confocal images of OPC processes in control- and Pcdh15 -shRNA cultures, treated with diluent (DMSO), CK666 (50 µM) or ML141 (10 µM), and immunolabelled to detect microtubules (α-tubulin, green) and F-actin (Phalloidin, red). n Phalloidin intensity (integrated pixel density) in the veils of OPCs from control- and Pcdh15 -shRNA cultures treated with DMSO, CK666 or ML141 [mean ± SEM, n = 3 independent cultures and ≥50 veils analysed per treatment condition; two‐way ANOVA: shRNA treatment F (1,12) = 5.22, p = 0.041; drug treatment F (2,12) = 86.54, p < 0.0001; interaction F (2,12) = 13.14, p = 0.0009 with Bonferroni multiple comparisons, * p < 0.01, **** p < 0.0001]. o Image series extracted from time-lapse videos depicting the filopodia of adjacent OPCs making contact in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141. Arrows indicate contacting filopodia. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight the formation and maintenance of filopodial contacts. p The mean duration that OPC filopodia remain in contact (min) in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥61 filopodial contacts analysed per treatment condition; two‐way ANOVA: shRNA treatment F (1,8) = 63.09, p < 0.0001; drug treatment F (1,8) = 135.0, p < 0.0001; interaction F (1,8) = 40.95, p < 0.0001 with Bonferroni multiple comparisons, * p < 0.05, **** p < 0.0001]. q The mean time taken for an OPC process to extrude and retract a veil in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥122 veils quantified per treatment condition; two‐way ANOVA: shRNA treatment F (1,8) = 47.69, p = 0.0001; drug treatment F (1,8) = 84.96, p < 0.0001; interaction F (1,8) = 21.94, p = 0.0016 with Bonferroni multiple comparisons, * p < 0.05, *** p < 0.001, **** p < 0.0001]. r Image series selected from time-lapse videos of control- and Pcdh15 -shRNA OPCs treated with DMSO or ML141, showing OPC processes as they extrude and retract veils. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight veil extrusion and retraction. Arrows indicate elaborated veils. Scale bars represent 2 µm ( b – m ) or 5 µm ( o , r ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a A schematic proposing a mechanism by which Pcdh15 could impact actin polymerisation in OPCs and depicting the point of action in the pathway of the drugs ML141 and CK666. b – m Confocal images of OPC processes in control- and Pcdh15 -shRNA cultures, treated with diluent (DMSO), CK666 (50 µM) or ML141 (10 µM), and immunolabelled to detect microtubules (α-tubulin, green) and F-actin (Phalloidin, red). n Phalloidin intensity (integrated pixel density) in the veils of OPCs from control- and Pcdh15 -shRNA cultures treated with DMSO, CK666 or ML141 [mean ± SEM, n = 3 independent cultures and ≥50 veils analysed per treatment condition; two‐way ANOVA: shRNA treatment F (1,12) = 5.22, p = 0.041; drug treatment F (2,12) = 86.54, p < 0.0001; interaction F (2,12) = 13.14, p = 0.0009 with Bonferroni multiple comparisons, * p < 0.01, **** p < 0.0001]. o Image series extracted from time-lapse videos depicting the filopodia of adjacent OPCs making contact in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141. Arrows indicate contacting filopodia. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight the formation and maintenance of filopodial contacts. p The mean duration that OPC filopodia remain in contact (min) in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥61 filopodial contacts analysed per treatment condition; two‐way ANOVA: shRNA treatment F (1,8) = 63.09, p < 0.0001; drug treatment F (1,8) = 135.0, p < 0.0001; interaction F (1,8) = 40.95, p < 0.0001 with Bonferroni multiple comparisons, * p < 0.05, **** p < 0.0001]. q The mean time taken for an OPC process to extrude and retract a veil in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥122 veils quantified per treatment condition; two‐way ANOVA: shRNA treatment F (1,8) = 47.69, p = 0.0001; drug treatment F (1,8) = 84.96, p < 0.0001; interaction F (1,8) = 21.94, p = 0.0016 with Bonferroni multiple comparisons, * p < 0.05, *** p < 0.001, **** p < 0.0001]. r Image series selected from time-lapse videos of control- and Pcdh15 -shRNA OPCs treated with DMSO or ML141, showing OPC processes as they extrude and retract veils. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight veil extrusion and retraction. Arrows indicate elaborated veils. Scale bars represent 2 µm ( b – m ) or 5 µm ( o , r ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: shRNA

    a Image series extracted from time-lapse videos to show individual control and Pcdh15 knockdown OPCs treated with DMSO or ML141 (10 µM), as they elaborate new processes over time. All images are time-stamped relative to the first image in the depicted series (designated as 0 min). White asterisks indicate the new process. b The mean number of processes supported by control and Pcdh15 knockdown OPCs treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥76 OPCs analysed per condition; one-way ANOVA: F (3,8) = 108.4, **** p < 0.0001]. c The mean number of new processes that OPCs elaborated per hour in control and Pcdh15 knockdown cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures and n ≥ 34 cells analysed per culture condition; two‐way ANOVA: shRNA treatment F (1,8) = 13.37, p = 0.0064; drug treatment F (1,8) = 35.06, p = 0.0004; interaction F (1,8) = 51.65, p < 0.0001 with Bonferroni multiple comparisons, *** p < 0.001]. Scale bars represent 10 µm.

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Image series extracted from time-lapse videos to show individual control and Pcdh15 knockdown OPCs treated with DMSO or ML141 (10 µM), as they elaborate new processes over time. All images are time-stamped relative to the first image in the depicted series (designated as 0 min). White asterisks indicate the new process. b The mean number of processes supported by control and Pcdh15 knockdown OPCs treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥76 OPCs analysed per condition; one-way ANOVA: F (3,8) = 108.4, **** p < 0.0001]. c The mean number of new processes that OPCs elaborated per hour in control and Pcdh15 knockdown cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures and n ≥ 34 cells analysed per culture condition; two‐way ANOVA: shRNA treatment F (1,8) = 13.37, p = 0.0064; drug treatment F (1,8) = 35.06, p = 0.0004; interaction F (1,8) = 51.65, p < 0.0001 with Bonferroni multiple comparisons, *** p < 0.001]. Scale bars represent 10 µm.

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: shRNA

    a – c Transverse spinal cord sections from embryonic day 16.5 (E16.5) mice were used to perform in situ hybridization to detect Pdgfrα or Pcdh15 mRNA. a E16.5 mouse spinal cord shows no labelling in the absence of an RNA probe. Line denotes the border of the spinal cord grey and white matter. b Pdgfrα + OPCs are readily detected in the E16.5 spinal cord white matter. The dashed box denotes the white matter region enlarged in the lower right corner. c Image showing Pcdh15 + cells in the E16.5 mouse spinal cord. The dashed box denotes the white matter region enlarged in the lower right corner. d Quantification of Pcdh15 mRNA (in FPKM, Fragments Per Kilobase Million) in neurons and glial cells acutely purified from the early postnatal mouse brain. Graph generated using previously published and publicly available RNA sequencing data from Zhang et al. . e Genome browser tracks of the Pcdh15 locus showing RNA-Seq alignments from nuclear RNA purified from the brains of adult Ng2-CreER T2 :: Sun1-eGFP or PLP-CreERT:: Sun1-eGFP mice. Pcdh15 mRNA is highly expressed by Ng2 + OPCs and most transcripts correspond to the CD3 isoforms of Pcdh15. f – h Immunocytochemistry to detect GFP (blue), PDGFRα (green) and Pcdh15 (red) in primary OPCs generated from the early postnatal Pdgfrα-histGFP mouse cortex. Scale bars represent 60 µm ( a – c ) and 20 µm ( f – h ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a – c Transverse spinal cord sections from embryonic day 16.5 (E16.5) mice were used to perform in situ hybridization to detect Pdgfrα or Pcdh15 mRNA. a E16.5 mouse spinal cord shows no labelling in the absence of an RNA probe. Line denotes the border of the spinal cord grey and white matter. b Pdgfrα + OPCs are readily detected in the E16.5 spinal cord white matter. The dashed box denotes the white matter region enlarged in the lower right corner. c Image showing Pcdh15 + cells in the E16.5 mouse spinal cord. The dashed box denotes the white matter region enlarged in the lower right corner. d Quantification of Pcdh15 mRNA (in FPKM, Fragments Per Kilobase Million) in neurons and glial cells acutely purified from the early postnatal mouse brain. Graph generated using previously published and publicly available RNA sequencing data from Zhang et al. . e Genome browser tracks of the Pcdh15 locus showing RNA-Seq alignments from nuclear RNA purified from the brains of adult Ng2-CreER T2 :: Sun1-eGFP or PLP-CreERT:: Sun1-eGFP mice. Pcdh15 mRNA is highly expressed by Ng2 + OPCs and most transcripts correspond to the CD3 isoforms of Pcdh15. f – h Immunocytochemistry to detect GFP (blue), PDGFRα (green) and Pcdh15 (red) in primary OPCs generated from the early postnatal Pdgfrα-histGFP mouse cortex. Scale bars represent 60 µm ( a – c ) and 20 µm ( f – h ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: In Situ Hybridization, Purification, Generated, RNA Sequencing Assay, Immunocytochemistry

    a Western blot gel image showing Pcdh15 expression in protein lysates generated from adult mouse brain ( n = 3 mice). b Western blot gel image showing control- and Pcdh15-shRNA treated 9DIV OPC cultures, 12 h after transfection. c Expression (pixel integrated density) of the ~62 KDa Pcdh15 variant was quantified relative to the expression of β-actin in protein lysates from control and Pcdh15 knockdown OPCs, 12 h after transfection. Data are normalised to the relative expression of Pcdh15 in control OPCs for each culture [mean ± SEM, n = 4 independent cultures; one sample t -test for Pcdh15-shRNA: deviation from 100 (%) = −96.02, SD of deviation = 2.54, t (3) = 75.33, p < 0.0001]. d – f Confocal images of control- and Pcdh15 -shRNA treated and untransfected OPCs following immunocytochemistry to detect Pcdh15 (red) and GFP (OPCs, green). g Quantification of Pcdh15 protein expression (immunocytochemistry pixel integrated density) in control- and Pcdh15-shRNA treated and untransfected OPCs [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (2,6) = 33.83, p = 0.0005; Tukey’s multiple comparisons test: *** p = 0.001, control shRNA vs. no transfection p = 0.99]. h – k Confocal images showing OPCs transfected with control shRNA or one of three distinct Pcdh15-shRNAs (Pcdh15-shRNA-A or Pcdh15-shRNA-B or Pcdh15-shRNA-C) for 12 h, before immunocytochemistry was performed to detect Pcdh15 (red) and GFP (OPCs, green). l Quantification of Pcdh15 expression (integrated pixel density) for OPCs transfected with control shRNA, Pcdh15-shRNA-A, Pcdh15-shRNA-B or Pcdh15-shRNA-C [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (3,8) = 31.05, p < 0.0001; Tukey’s multiple comparisons test: *** p < 0.001; Pcdh15-shRNA-A vs. B and B vs. C, p = 0.9; Pcdh15-shRNA-A vs. C p = 0.6]. Scale bars represent 30 µm ( d – f ) or 20 µm ( h – k ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Western blot gel image showing Pcdh15 expression in protein lysates generated from adult mouse brain ( n = 3 mice). b Western blot gel image showing control- and Pcdh15-shRNA treated 9DIV OPC cultures, 12 h after transfection. c Expression (pixel integrated density) of the ~62 KDa Pcdh15 variant was quantified relative to the expression of β-actin in protein lysates from control and Pcdh15 knockdown OPCs, 12 h after transfection. Data are normalised to the relative expression of Pcdh15 in control OPCs for each culture [mean ± SEM, n = 4 independent cultures; one sample t -test for Pcdh15-shRNA: deviation from 100 (%) = −96.02, SD of deviation = 2.54, t (3) = 75.33, p < 0.0001]. d – f Confocal images of control- and Pcdh15 -shRNA treated and untransfected OPCs following immunocytochemistry to detect Pcdh15 (red) and GFP (OPCs, green). g Quantification of Pcdh15 protein expression (immunocytochemistry pixel integrated density) in control- and Pcdh15-shRNA treated and untransfected OPCs [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (2,6) = 33.83, p = 0.0005; Tukey’s multiple comparisons test: *** p = 0.001, control shRNA vs. no transfection p = 0.99]. h – k Confocal images showing OPCs transfected with control shRNA or one of three distinct Pcdh15-shRNAs (Pcdh15-shRNA-A or Pcdh15-shRNA-B or Pcdh15-shRNA-C) for 12 h, before immunocytochemistry was performed to detect Pcdh15 (red) and GFP (OPCs, green). l Quantification of Pcdh15 expression (integrated pixel density) for OPCs transfected with control shRNA, Pcdh15-shRNA-A, Pcdh15-shRNA-B or Pcdh15-shRNA-C [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (3,8) = 31.05, p < 0.0001; Tukey’s multiple comparisons test: *** p < 0.001; Pcdh15-shRNA-A vs. B and B vs. C, p = 0.9; Pcdh15-shRNA-A vs. C p = 0.6]. Scale bars represent 30 µm ( d – f ) or 20 µm ( h – k ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Western Blot, Expressing, Generated, shRNA, Transfection, Variant Assay, Immunocytochemistry

    a – f Confocal images showing control ( a – c ) or Pcdh15 knockdown ( d – f ) GFP + OPCs (green) after 12 h of EdU labelling (red). White arrowheads indicate EdU + GFP + OPCs. g The proportion of control and Pcdh15 knockdown GFP + OPCs (%) that incorporated EdU (EdU + GFP + /GFP + × 100; mean ± SEM, n = 3 independent cultures; unpaired t -test, * p = 0.048). h The proportion (%) of GFP + OPCs that incorporate EdU (EdU + GFP + /GFP + × 100) in cultures transfected with control-shRNA, Pcdh15-shRNA-A, Pcdh15-shRNA-B or Pcdh15-shRNA-C [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (3,8) = 5.658, p = 0.02; Tukey’s multiple comparisons test: Control shRNA vs Pch15 shRNA-A, * p < 0.05; Pcdh15-shRNA-A vs. Pcdh15-shRNA-B, p = 1.0; Pcdh15-shRNA-A vs. Pcdh15-shRNA-C, p = 1.0, Pcdh15-shRNA-B vs. Pcdh15-shRNA-C, p = 0.99]. i – p Confocal images showing EdU-labelling (red) in GFP + OPCs (green) transfected with control-shRNA ( i , j ), Pcdh15-shRNA-A ( k , l ), Pcdh15-shRNA-B ( m , n ) or Pcdh15-shRNA-C ( o , p ). q The proportion (%) of EdU + GFP + nuclei that belonged to: single cells (nuclei not contacting others); paired cells (two nuclei in contact), or clustered cells (groups of three or four nuclei in contact with each other) [mean ± SEM for n = 3 independent cultures; ≥86 EdU + GFP + cells analysed per coverslip. Two-way ANOVA: shRNA F (1, 16) = 0.0037, p = 0.9524; cluster F (3, 16) = 6206, p < 0.0001; interaction F (3, 16) = 35.79, p < 0.0001; with Bonferroni multiple comparison test: **** p < 0.0001. r Quantification of the mean Euclidean distance between EdU + GFP + nuclei (mean ± SEM, n = 3 independent cultures; ≥86 EdU + GFP + cells analysed per coverslip, unpaired t -test with welch’s correction, * p = 0.024). Scale bars represent 25 µm ( a – f ) or 15 µm ( i – l ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a – f Confocal images showing control ( a – c ) or Pcdh15 knockdown ( d – f ) GFP + OPCs (green) after 12 h of EdU labelling (red). White arrowheads indicate EdU + GFP + OPCs. g The proportion of control and Pcdh15 knockdown GFP + OPCs (%) that incorporated EdU (EdU + GFP + /GFP + × 100; mean ± SEM, n = 3 independent cultures; unpaired t -test, * p = 0.048). h The proportion (%) of GFP + OPCs that incorporate EdU (EdU + GFP + /GFP + × 100) in cultures transfected with control-shRNA, Pcdh15-shRNA-A, Pcdh15-shRNA-B or Pcdh15-shRNA-C [mean ± SEM, n = 3 independent cultures; one‐way ANOVA: F (3,8) = 5.658, p = 0.02; Tukey’s multiple comparisons test: Control shRNA vs Pch15 shRNA-A, * p < 0.05; Pcdh15-shRNA-A vs. Pcdh15-shRNA-B, p = 1.0; Pcdh15-shRNA-A vs. Pcdh15-shRNA-C, p = 1.0, Pcdh15-shRNA-B vs. Pcdh15-shRNA-C, p = 0.99]. i – p Confocal images showing EdU-labelling (red) in GFP + OPCs (green) transfected with control-shRNA ( i , j ), Pcdh15-shRNA-A ( k , l ), Pcdh15-shRNA-B ( m , n ) or Pcdh15-shRNA-C ( o , p ). q The proportion (%) of EdU + GFP + nuclei that belonged to: single cells (nuclei not contacting others); paired cells (two nuclei in contact), or clustered cells (groups of three or four nuclei in contact with each other) [mean ± SEM for n = 3 independent cultures; ≥86 EdU + GFP + cells analysed per coverslip. Two-way ANOVA: shRNA F (1, 16) = 0.0037, p = 0.9524; cluster F (3, 16) = 6206, p < 0.0001; interaction F (3, 16) = 35.79, p < 0.0001; with Bonferroni multiple comparison test: **** p < 0.0001. r Quantification of the mean Euclidean distance between EdU + GFP + nuclei (mean ± SEM, n = 3 independent cultures; ≥86 EdU + GFP + cells analysed per coverslip, unpaired t -test with welch’s correction, * p = 0.024). Scale bars represent 25 µm ( a – f ) or 15 µm ( i – l ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Transfection, shRNA

    a Primary mouse OPC cultures were transfected with control- or Pcdh15-shRNA and protein lysates collected 12 h later, at 10 DIV. Image of a Western blot performed to detect phosphorylated β-catenin (p-β-catenin), active unphosphorylated β-catenin, and β-actin. b Quantification of p-β-catenin (Ser33/Ser37/Thr41) expression by OPC cultures transfected with control- or Pcdh15-shRNA, normalised to β-actin expression (mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.17). c Quantification of β-catenin expression by OPC cultures, 12 h after transfection with control- or Pcdh15-shRNA, normalised to β-actin expression (mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.63). d Western blot gel image showing ERK1/2, phosphorylated ERK1/2, β-actin, and GAPDH expression by OPCs transfected with control- or Pcdh15-shRNA. e Western blot protein band integrated pixel density was quantified for p-ERK1/2 and ERK1/2 and used to calculate the proportion of ERK1 that was phosphorylated (p-ERK1/ERK1) and the proportion of ERK2 that was phosphorylated (p-ERK2/ERK2) for OPCs transfected with control- or Pcdh15-shRNA [mean ± SEM, n = 3 independent cultures; p-ERK1/ERK1 and p-ERK2/ERK2 data were analysed separately by unpaired t -test (note // break to x -axis), * p < 0.05, ** p < 0.01]. f Western blot gel image showing ERK1/2, p-ERK1/2, β-actin, and GAPDH expression by OPCs transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126 (10 µM). g Quantification of the proportion of ERK1 that was phosphorylated (integrated pixel density for p-ERK1/integrated pixel density for ERK1) and the proportion of ERK2 that was phosphorylated (integrated pixel density p-ERK2/integrated pixel density for ERK2) in OPCs transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126. For each culture, these data were normalised to the control shRNA group, i.e., relative protein level/control shRNA relative protein level × 100 [mean ± SEM, n = 3 independent cultures; two‐way ANOVA for p-ERK1/ERK1: shRNA treatment F (1,8) = 11.06, p = 0.0104; drug treatment F (1,8) = 107.9, p < 0.0001; interaction F (1,8) = 10.18, p = 0.012 with Bonferroni multiple comparisons, ** p < 0.01, **** p < 0.0001; two-way ANOVA for p-ERK2/ERK2: shRNA treatment F (1,8) = 2.12, p = 0.061; drug treatment F (1,8) = 91.19, p < 0.0001; interaction F (1,8) = 3.03, p = 0.032 with Bonferroni multiple comparisons, * p < 0.05, **** p < 0.0001]. h – k Confocal images showing GFP + OPCs (green) transfected with control- or Pcdh15 -shRNA, treated with DMSO or U0126 and exposed to EdU (red) for 12 h. l The proportion (%) of GFP + control or Pcdh15 knockdown OPCs that incorporate EdU following treatment with DMSO or U0126 [mean ± SEM, n = 3 independent cultures; two‐way ANOVA: shRNA F (1,8) = 13.0, p = 0.007; drug treatment F (1,8) = 62.98, p < 0.0001; interaction F (1,8) = 6.91, p = 0.03 with Bonferroni multiple comparisons test: * p < 0.05, *** p < 0.001]. Scale bars represent 35 µm.

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Primary mouse OPC cultures were transfected with control- or Pcdh15-shRNA and protein lysates collected 12 h later, at 10 DIV. Image of a Western blot performed to detect phosphorylated β-catenin (p-β-catenin), active unphosphorylated β-catenin, and β-actin. b Quantification of p-β-catenin (Ser33/Ser37/Thr41) expression by OPC cultures transfected with control- or Pcdh15-shRNA, normalised to β-actin expression (mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.17). c Quantification of β-catenin expression by OPC cultures, 12 h after transfection with control- or Pcdh15-shRNA, normalised to β-actin expression (mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.63). d Western blot gel image showing ERK1/2, phosphorylated ERK1/2, β-actin, and GAPDH expression by OPCs transfected with control- or Pcdh15-shRNA. e Western blot protein band integrated pixel density was quantified for p-ERK1/2 and ERK1/2 and used to calculate the proportion of ERK1 that was phosphorylated (p-ERK1/ERK1) and the proportion of ERK2 that was phosphorylated (p-ERK2/ERK2) for OPCs transfected with control- or Pcdh15-shRNA [mean ± SEM, n = 3 independent cultures; p-ERK1/ERK1 and p-ERK2/ERK2 data were analysed separately by unpaired t -test (note // break to x -axis), * p < 0.05, ** p < 0.01]. f Western blot gel image showing ERK1/2, p-ERK1/2, β-actin, and GAPDH expression by OPCs transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126 (10 µM). g Quantification of the proportion of ERK1 that was phosphorylated (integrated pixel density for p-ERK1/integrated pixel density for ERK1) and the proportion of ERK2 that was phosphorylated (integrated pixel density p-ERK2/integrated pixel density for ERK2) in OPCs transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126. For each culture, these data were normalised to the control shRNA group, i.e., relative protein level/control shRNA relative protein level × 100 [mean ± SEM, n = 3 independent cultures; two‐way ANOVA for p-ERK1/ERK1: shRNA treatment F (1,8) = 11.06, p = 0.0104; drug treatment F (1,8) = 107.9, p < 0.0001; interaction F (1,8) = 10.18, p = 0.012 with Bonferroni multiple comparisons, ** p < 0.01, **** p < 0.0001; two-way ANOVA for p-ERK2/ERK2: shRNA treatment F (1,8) = 2.12, p = 0.061; drug treatment F (1,8) = 91.19, p < 0.0001; interaction F (1,8) = 3.03, p = 0.032 with Bonferroni multiple comparisons, * p < 0.05, **** p < 0.0001]. h – k Confocal images showing GFP + OPCs (green) transfected with control- or Pcdh15 -shRNA, treated with DMSO or U0126 and exposed to EdU (red) for 12 h. l The proportion (%) of GFP + control or Pcdh15 knockdown OPCs that incorporate EdU following treatment with DMSO or U0126 [mean ± SEM, n = 3 independent cultures; two‐way ANOVA: shRNA F (1,8) = 13.0, p = 0.007; drug treatment F (1,8) = 62.98, p < 0.0001; interaction F (1,8) = 6.91, p = 0.03 with Bonferroni multiple comparisons test: * p < 0.05, *** p < 0.001]. Scale bars represent 35 µm.

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Transfection, shRNA, Western Blot, Expressing

    12 h after OPCs were transfected with control or Pcdh15 -shRNA, they were imaged once a minute for 2 h and the resulting time-lapse videos were used to quantify different aspects of OPC motility. a Image series show a filopodial contact (black arrowhead) between adjacent OPCs in a control and Pcdh15 knockdown culture. All images are time-stamped relative to the first image in the depicted series (designated as 0 min). b The mean duration (min) that a filopodial contact was sustained in control and Pcdh15 knockdown cultures (mean ± SEM, n = 3 independent cultures, ≥66 filopodial contacts measured per condition; unpaired t test, * p = 0.034). c Excepts from a time-lapse image series showing a control OPC process as it extrudes and retracts a veil and a Pcdh15 knockdown OPC as it extrudes and maintains its veil. The images are time-stamped relative to the first image in the depicted series (designated as 0 min). Black arrowheads indicate elaborated veils. d Quantification of the mean veiling time for OPC processes in control- and Pcdh15 -shRNA treated cultures (time taken to complete an extrusion and retraction) (mean ± SEM, n = 3 independent cultures, ≥146 veils analysed per condition; unpaired t -test, ** p = 0.004). e Images of control- and Pcdh15 -shRNA treated OPCs, 12 h after transfection. f Quantification of the mean number of processes supported by control and Pcdh15 shRNA-treated OPCs (mean ± SEM, n = 3 independent cultures and ≥81 OPCs analysed per condition; unpaired t test, *** p = 0.0004). g Short image series extracted from the time-lapse videos to show control and Pcdh15 knockdown OPCs extending a new process. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and the white asterisks denote the new processes. h Quantification of the mean number of new processes elaborated by control- and Pcdh15 -shRNA transfected OPCs each hour (mean ± SEM, n = 3 independent cultures and ≥35 OPCs analysed per condition; unpaired t test, * p = 0.048). i The migration trajectories for the soma of 52 control- or Pcdh15 -shRNA treated OPCs. j Mean migration speed for the soma of control- and Pcdh15 -shRNA treated OPCs (mean ± SEM, n = 52 OPCs per group, unpaired t tests with Welch’s correction, **** p < 0.0001). k Mean distance that each OPC soma moved (path length) in control- and Pcdh15 -shRNA treated cultures (mean ± SEM, n = 52 OPCs per group, unpaired t tests with Welch’s correction, **** p < 0.0001). l Mean Euclidean distance (shortest distance between the start and end points) that each OPC soma moved in control- or Pcdh15 -shRNA treated cultures (mean ± SEM, unpaired t tests with Welch’s correction, *** p < 0.0002). Scale bars represent 5 µm ( a , c ), 8 µm ( g ) or 20 µm ( e ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: 12 h after OPCs were transfected with control or Pcdh15 -shRNA, they were imaged once a minute for 2 h and the resulting time-lapse videos were used to quantify different aspects of OPC motility. a Image series show a filopodial contact (black arrowhead) between adjacent OPCs in a control and Pcdh15 knockdown culture. All images are time-stamped relative to the first image in the depicted series (designated as 0 min). b The mean duration (min) that a filopodial contact was sustained in control and Pcdh15 knockdown cultures (mean ± SEM, n = 3 independent cultures, ≥66 filopodial contacts measured per condition; unpaired t test, * p = 0.034). c Excepts from a time-lapse image series showing a control OPC process as it extrudes and retracts a veil and a Pcdh15 knockdown OPC as it extrudes and maintains its veil. The images are time-stamped relative to the first image in the depicted series (designated as 0 min). Black arrowheads indicate elaborated veils. d Quantification of the mean veiling time for OPC processes in control- and Pcdh15 -shRNA treated cultures (time taken to complete an extrusion and retraction) (mean ± SEM, n = 3 independent cultures, ≥146 veils analysed per condition; unpaired t -test, ** p = 0.004). e Images of control- and Pcdh15 -shRNA treated OPCs, 12 h after transfection. f Quantification of the mean number of processes supported by control and Pcdh15 shRNA-treated OPCs (mean ± SEM, n = 3 independent cultures and ≥81 OPCs analysed per condition; unpaired t test, *** p = 0.0004). g Short image series extracted from the time-lapse videos to show control and Pcdh15 knockdown OPCs extending a new process. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and the white asterisks denote the new processes. h Quantification of the mean number of new processes elaborated by control- and Pcdh15 -shRNA transfected OPCs each hour (mean ± SEM, n = 3 independent cultures and ≥35 OPCs analysed per condition; unpaired t test, * p = 0.048). i The migration trajectories for the soma of 52 control- or Pcdh15 -shRNA treated OPCs. j Mean migration speed for the soma of control- and Pcdh15 -shRNA treated OPCs (mean ± SEM, n = 52 OPCs per group, unpaired t tests with Welch’s correction, **** p < 0.0001). k Mean distance that each OPC soma moved (path length) in control- and Pcdh15 -shRNA treated cultures (mean ± SEM, n = 52 OPCs per group, unpaired t tests with Welch’s correction, **** p < 0.0001). l Mean Euclidean distance (shortest distance between the start and end points) that each OPC soma moved in control- or Pcdh15 -shRNA treated cultures (mean ± SEM, unpaired t tests with Welch’s correction, *** p < 0.0002). Scale bars represent 5 µm ( a , c ), 8 µm ( g ) or 20 µm ( e ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Transfection, shRNA, Migration

    a Image series extracted from time-lapse videos showing filopodial contacts (arrows) formed between adjacent OPCs in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126 (10 µM). All images are time-stamped relative to the first image in the depicted series (designated as 0 min). b The mean duration (min) that filopodial contacts were sustained once they formed between adjacent OPCs in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥44 filopodial contacts measured per condition; two‐way ANOVA: shRNA treatment F (1,8) = 56.29, p < 0.0001; drug treatment F (1,8) = 0.0015, p = 0.96; interaction F (1,8) = 0.00017, p = 0.98 with Bonferroni multiple comparisons test: ** p < 0.01]. c Mean OPC process veiling time (min), i.e., the time taken for an OPC process to complete one full extrusion and retraction of its veil in control- or Pcdh15 -shRNA cultures treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥102 veils analysed per condition; two‐way ANOVA: shRNA treatment F (1,8) = 52.44, p < 0.0001; drug treatment F (1,8) = 0.36, p = 0.56; interaction F (1,8) = 1.05, p = 0.33 with Bonferroni multiple comparisons, ** p < 0.01, *** p < 0.001]. d Image series extracted from time-lapse videos showing OPC processes elaborating and retracting veils in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126. White arrows indicate extruded veils. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight veil extrusion and retraction. e Images of OPCs 12 h after transfection with control- or Pcdh15-shRNA and treatment with DMSO or U0126 (10 µM). f Quantification of the number of new processes elaborated by OPCs each hour in cultures transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥31 OPCs analysed per condition; two‐way ANOVA: shRNA treatment F (1,8) = 314.7, p < 0.0001; drug treatment F (1,8) = 0.48, p = 0.50; interaction F (1,8) = 0.0007, p = 0.97 with Bonferroni multiple comparisons, **** p < 0.0001]. Image series depicting OPC process generation can be found in Supplementary Fig. . Scale bars represent 5 µm ( a , d ) or 10 µm ( e ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Image series extracted from time-lapse videos showing filopodial contacts (arrows) formed between adjacent OPCs in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126 (10 µM). All images are time-stamped relative to the first image in the depicted series (designated as 0 min). b The mean duration (min) that filopodial contacts were sustained once they formed between adjacent OPCs in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥44 filopodial contacts measured per condition; two‐way ANOVA: shRNA treatment F (1,8) = 56.29, p < 0.0001; drug treatment F (1,8) = 0.0015, p = 0.96; interaction F (1,8) = 0.00017, p = 0.98 with Bonferroni multiple comparisons test: ** p < 0.01]. c Mean OPC process veiling time (min), i.e., the time taken for an OPC process to complete one full extrusion and retraction of its veil in control- or Pcdh15 -shRNA cultures treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥102 veils analysed per condition; two‐way ANOVA: shRNA treatment F (1,8) = 52.44, p < 0.0001; drug treatment F (1,8) = 0.36, p = 0.56; interaction F (1,8) = 1.05, p = 0.33 with Bonferroni multiple comparisons, ** p < 0.01, *** p < 0.001]. d Image series extracted from time-lapse videos showing OPC processes elaborating and retracting veils in control- and Pcdh15 -shRNA cultures treated with DMSO or U0126. White arrows indicate extruded veils. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight veil extrusion and retraction. e Images of OPCs 12 h after transfection with control- or Pcdh15-shRNA and treatment with DMSO or U0126 (10 µM). f Quantification of the number of new processes elaborated by OPCs each hour in cultures transfected with control- or Pcdh15-shRNA and treated with DMSO or U0126 [mean ± SEM, n = 3 independent cultures, ≥31 OPCs analysed per condition; two‐way ANOVA: shRNA treatment F (1,8) = 314.7, p < 0.0001; drug treatment F (1,8) = 0.48, p = 0.50; interaction F (1,8) = 0.0007, p = 0.97 with Bonferroni multiple comparisons, **** p < 0.0001]. Image series depicting OPC process generation can be found in Supplementary Fig. . Scale bars represent 5 µm ( a , d ) or 10 µm ( e ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: shRNA, Transfection

    a Confocal images of OPC processes in control- and Pcdh15 shRNA treated cultures immunolabelled to detect microtubules (α-tubulin, green) and F-actin (Phalloidin, red). b Quantification of phalloidin intensity (integrated pixel density) in control and Pcdh15-knockdown OPCs (normalized to the average intensity of control OPC cultures) ( n = 3 independent cultures, ≥51 veils analysed per condition; one-sample t -test for Pcdh15-shRNA: deviation from 100(%) = 30.25, SD of deviation = 9.19, t (2) = 5.697, p = 0.029). c Degree of colocalization between actin (phalloidin) and microtubule (α-actin) labelling in control and Pcdh15 knockdown OPC veils. Colocalization is expressed as a Pearson correlation coefficient (mean ± SEM, n = 3 independent cultures, ≥37 veils measured per condition; unpaired t -test, p = 0.81). d Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect Akt, phosphorylated Akt (p-Akt) and β-actin. e Expression of Akt in control and Pcdh15 knockdown OPC cultures, normalised to β-actin expression (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.12). f Quantification of p-Akt (Ser473) and Akt in control and Pcdh15 knockdown OPCs, normalised to β-actin (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.20). g Average fold change in cytoskeletal gene expression between OPCs transfected with control- and Pcdh15 -shRNA (average from n = 3 independent cultures). h Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect CrkII and WAVE2, which are upstream of the Arp2/3 complex, Arp2, Arp3 and Arpc3, which are components of the Arp2/3 complex, and GAPDH. i CrkII, j WAVE2, k Arp2, l Arp3, and m Arpc3 protein expression was quantified by measuring the integrated pixel density of the corresponding Western blot band and normalising expression to GAPDH expression for each sample [mean ± SEM, n = 3 independent cultures, expression in control and Pcdh15 knockdown OPCs was compared by unpaired t -tests: i p = 0.039, j p = 0.004, k p = 0.0001, l p = 0.028, m p = 0.016]. Scale bars represent 5 µm.

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Confocal images of OPC processes in control- and Pcdh15 shRNA treated cultures immunolabelled to detect microtubules (α-tubulin, green) and F-actin (Phalloidin, red). b Quantification of phalloidin intensity (integrated pixel density) in control and Pcdh15-knockdown OPCs (normalized to the average intensity of control OPC cultures) ( n = 3 independent cultures, ≥51 veils analysed per condition; one-sample t -test for Pcdh15-shRNA: deviation from 100(%) = 30.25, SD of deviation = 9.19, t (2) = 5.697, p = 0.029). c Degree of colocalization between actin (phalloidin) and microtubule (α-actin) labelling in control and Pcdh15 knockdown OPC veils. Colocalization is expressed as a Pearson correlation coefficient (mean ± SEM, n = 3 independent cultures, ≥37 veils measured per condition; unpaired t -test, p = 0.81). d Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect Akt, phosphorylated Akt (p-Akt) and β-actin. e Expression of Akt in control and Pcdh15 knockdown OPC cultures, normalised to β-actin expression (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.12). f Quantification of p-Akt (Ser473) and Akt in control and Pcdh15 knockdown OPCs, normalised to β-actin (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t -test, p = 0.20). g Average fold change in cytoskeletal gene expression between OPCs transfected with control- and Pcdh15 -shRNA (average from n = 3 independent cultures). h Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect CrkII and WAVE2, which are upstream of the Arp2/3 complex, Arp2, Arp3 and Arpc3, which are components of the Arp2/3 complex, and GAPDH. i CrkII, j WAVE2, k Arp2, l Arp3, and m Arpc3 protein expression was quantified by measuring the integrated pixel density of the corresponding Western blot band and normalising expression to GAPDH expression for each sample [mean ± SEM, n = 3 independent cultures, expression in control and Pcdh15 knockdown OPCs was compared by unpaired t -tests: i p = 0.039, j p = 0.004, k p = 0.0001, l p = 0.028, m p = 0.016]. Scale bars represent 5 µm.

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: shRNA, Western Blot, Expressing, Transfection

    a – c Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and Hoechst 33342 (HST; blue). d Single confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and Pcdh15 (green). e – g Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and HST (blue). h Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and Pcdh15 (green). i – k Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red) and the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). l Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and the CD3 isoforms of Pcdh15 (green). (m-o) Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). p Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and the CD3 isoforms of Pcdh15 (green). Scale bars represent 20 µm ( a – c , e – g , i – k , m – o ) or 5 µm ( d , h , l , p ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a – c Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and Hoechst 33342 (HST; blue). d Single confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and Pcdh15 (green). e – g Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and HST (blue). h Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and Pcdh15 (green). i – k Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red) and the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). l Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and the CD3 isoforms of Pcdh15 (green). (m-o) Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). p Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and the CD3 isoforms of Pcdh15 (green). Scale bars represent 20 µm ( a – c , e – g , i – k , m – o ) or 5 µm ( d , h , l , p ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: Expressing

    a A schematic proposing a mechanism by which Pcdh15 could impact actin polymerisation in OPCs and depicting the point of action in the pathway of the drugs ML141 and CK666. b – m Confocal images of OPC processes in control- and Pcdh15 -shRNA cultures, treated with diluent (DMSO), CK666 (50 µM) or ML141 (10 µM), and immunolabelled to detect microtubules (α-tubulin, green) and F-actin (Phalloidin, red). n Phalloidin intensity (integrated pixel density) in the veils of OPCs from control- and Pcdh15 -shRNA cultures treated with DMSO, CK666 or ML141 [mean ± SEM, n = 3 independent cultures and ≥50 veils analysed per treatment condition; two‐way ANOVA: shRNA treatment F (1,12) = 5.22, p = 0.041; drug treatment F (2,12) = 86.54, p < 0.0001; interaction F (2,12) = 13.14, p = 0.0009 with Bonferroni multiple comparisons, * p < 0.01, **** p < 0.0001]. o Image series extracted from time-lapse videos depicting the filopodia of adjacent OPCs making contact in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141. Arrows indicate contacting filopodia. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight the formation and maintenance of filopodial contacts. p The mean duration that OPC filopodia remain in contact (min) in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥61 filopodial contacts analysed per treatment condition; two‐way ANOVA: shRNA treatment F (1,8) = 63.09, p < 0.0001; drug treatment F (1,8) = 135.0, p < 0.0001; interaction F (1,8) = 40.95, p < 0.0001 with Bonferroni multiple comparisons, * p < 0.05, **** p < 0.0001]. q The mean time taken for an OPC process to extrude and retract a veil in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥122 veils quantified per treatment condition; two‐way ANOVA: shRNA treatment F (1,8) = 47.69, p = 0.0001; drug treatment F (1,8) = 84.96, p < 0.0001; interaction F (1,8) = 21.94, p = 0.0016 with Bonferroni multiple comparisons, * p < 0.05, *** p < 0.001, **** p < 0.0001]. r Image series selected from time-lapse videos of control- and Pcdh15 -shRNA OPCs treated with DMSO or ML141, showing OPC processes as they extrude and retract veils. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight veil extrusion and retraction. Arrows indicate elaborated veils. Scale bars represent 2 µm ( b – m ) or 5 µm ( o , r ).

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a A schematic proposing a mechanism by which Pcdh15 could impact actin polymerisation in OPCs and depicting the point of action in the pathway of the drugs ML141 and CK666. b – m Confocal images of OPC processes in control- and Pcdh15 -shRNA cultures, treated with diluent (DMSO), CK666 (50 µM) or ML141 (10 µM), and immunolabelled to detect microtubules (α-tubulin, green) and F-actin (Phalloidin, red). n Phalloidin intensity (integrated pixel density) in the veils of OPCs from control- and Pcdh15 -shRNA cultures treated with DMSO, CK666 or ML141 [mean ± SEM, n = 3 independent cultures and ≥50 veils analysed per treatment condition; two‐way ANOVA: shRNA treatment F (1,12) = 5.22, p = 0.041; drug treatment F (2,12) = 86.54, p < 0.0001; interaction F (2,12) = 13.14, p = 0.0009 with Bonferroni multiple comparisons, * p < 0.01, **** p < 0.0001]. o Image series extracted from time-lapse videos depicting the filopodia of adjacent OPCs making contact in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141. Arrows indicate contacting filopodia. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight the formation and maintenance of filopodial contacts. p The mean duration that OPC filopodia remain in contact (min) in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥61 filopodial contacts analysed per treatment condition; two‐way ANOVA: shRNA treatment F (1,8) = 63.09, p < 0.0001; drug treatment F (1,8) = 135.0, p < 0.0001; interaction F (1,8) = 40.95, p < 0.0001 with Bonferroni multiple comparisons, * p < 0.05, **** p < 0.0001]. q The mean time taken for an OPC process to extrude and retract a veil in control- and Pcdh15 -shRNA cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥122 veils quantified per treatment condition; two‐way ANOVA: shRNA treatment F (1,8) = 47.69, p = 0.0001; drug treatment F (1,8) = 84.96, p < 0.0001; interaction F (1,8) = 21.94, p = 0.0016 with Bonferroni multiple comparisons, * p < 0.05, *** p < 0.001, **** p < 0.0001]. r Image series selected from time-lapse videos of control- and Pcdh15 -shRNA OPCs treated with DMSO or ML141, showing OPC processes as they extrude and retract veils. All images are time-stamped relative to the first image in the depicted series (designated as 0 min) and images are selected to highlight veil extrusion and retraction. Arrows indicate elaborated veils. Scale bars represent 2 µm ( b – m ) or 5 µm ( o , r ).

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: shRNA

    a Image series extracted from time-lapse videos to show individual control and Pcdh15 knockdown OPCs treated with DMSO or ML141 (10 µM), as they elaborate new processes over time. All images are time-stamped relative to the first image in the depicted series (designated as 0 min). White asterisks indicate the new process. b The mean number of processes supported by control and Pcdh15 knockdown OPCs treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥76 OPCs analysed per condition; one-way ANOVA: F (3,8) = 108.4, **** p < 0.0001]. c The mean number of new processes that OPCs elaborated per hour in control and Pcdh15 knockdown cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures and n ≥ 34 cells analysed per culture condition; two‐way ANOVA: shRNA treatment F (1,8) = 13.37, p = 0.0064; drug treatment F (1,8) = 35.06, p = 0.0004; interaction F (1,8) = 51.65, p < 0.0001 with Bonferroni multiple comparisons, *** p < 0.001]. Scale bars represent 10 µm.

    Journal: Communications Biology

    Article Title: Protocadherin 15 suppresses oligodendrocyte progenitor cell proliferation and promotes motility through distinct signalling pathways

    doi: 10.1038/s42003-022-03470-1

    Figure Lengend Snippet: a Image series extracted from time-lapse videos to show individual control and Pcdh15 knockdown OPCs treated with DMSO or ML141 (10 µM), as they elaborate new processes over time. All images are time-stamped relative to the first image in the depicted series (designated as 0 min). White asterisks indicate the new process. b The mean number of processes supported by control and Pcdh15 knockdown OPCs treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures, ≥76 OPCs analysed per condition; one-way ANOVA: F (3,8) = 108.4, **** p < 0.0001]. c The mean number of new processes that OPCs elaborated per hour in control and Pcdh15 knockdown cultures treated with DMSO or ML141 [mean ± SEM, n = 3 independent cultures and n ≥ 34 cells analysed per culture condition; two‐way ANOVA: shRNA treatment F (1,8) = 13.37, p = 0.0064; drug treatment F (1,8) = 35.06, p = 0.0004; interaction F (1,8) = 51.65, p < 0.0001 with Bonferroni multiple comparisons, *** p < 0.001]. Scale bars represent 10 µm.

    Article Snippet: OPC complete culture medium was replaced with OPC complete culture medium containing 5 µg/ml polybrene (Santa Cruz Biotechnology, sc-132220) and the relevant shRNA lentiviral particles: control shRNA lentiviral particles-A (Santa Cruz Biotechnology, sc-108080) or Pcdh15 shRNA lentiviral particles (Santa Cruz Biotechnology, sc-152056-V), which contained a mixture of: Pcdh15 shRNA-A (sc-152056-VA: GATCC GGATA AGACT CGCTA CTATT TCAAG AGAAT AGTAG CGAGT CTTAT CCTTT TT); Pcdh15 shRNA-B (sc-152056-VB: GATCC GTCTG CACAT CGAAA TACTT TCAAG AGAAG TATTT CGATG TGCAG ACTTT TT) and Pcdh15 shRNA-C (sc-152056-VC: GATCC GACTA TGCCA CCTGG TATAT TCAAG AGATA TACCA GGTGG CATAG TCTTT TT).

    Techniques: shRNA