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