Fig 1: Distinct phenotypes of prion-modulatory proteins at the ECM of chronically infected cellsA–D Chronically infected iS7 cells were fixed, delipidated with acetone, and denatured with 3 M GTC. Cells were then co-labelled with ICSM18 and (A) anti-Fn1, (B) anti-Chga, (C) anti-Lrrn4, and (D) anti-Itga8. After washing of primary antibodies with sterile PBS, cells were incubated with highly cross-absorbed anti-mouse (ICSM18) and anti-rabbit (all other antibodies) Alexa Fluor-conjugated secondary antibodies. Representative images are shown. PCC values: (A) 0.03 ± 0.03, (B) 0.08 ± 0.03, (C) 0.16 ± 0.09 and (D) 0.28 ± 0.07.Data information: Scale bar, 20 μm.
Fig 2: Effects of CgA in cell proliferation and phenotypic changes in additional neuroblastoma cell lines. (A) CgA mRNA expression was evaluated in a series of neuroblastoma cell lines with (BE(2)-M17 and IMR-32) and without (SH-SY5Y and SK-N-SH) N-Myc amplification. Relative CgA mRNA expression was calculated using the 2-??CT method normalized to that in SH-SY5Y cells. (B) SiRNA CgA and SiRNA control were transfected into BE(2)-M17 and hCgA-pCMV6-Entry plasmid and empty vector were transfected in SK-N-SH and IMR-32 cells for knockdown and overexpression experiments respectively. 24 h later, the cells were collected to analyze CgA expression by real-time PCR. (C) The effects of CgA knockdown and overexpression in proliferation rates in BE(2)-M17, SK-N-SH and IMR-32 cells were measured by BrdU incorporation assay. (D–F) Cell linage specific markers were examined following CgA knockdown in BE(2)-M17 cells (D), CgA overexpression in SK-N-SH (E) and IMR-32 (F) cells by real-time PCR. Normalization over siRNA control or vector control was used to calculate fold changes (B–F). Each bar indicates the mean±s.d. of triplicate tests. Data were analyzed by two-tailed unpaired t-test with Welch's correction, *P<0.05; **P<0.01; ***P<0.005.
Fig 3: ShRNA-directed CgA depletion promotes Schwann cell differentiation in human neuroblastoma SH-SY5Y cells. (A) Quantitative PCR (qPCR) results depicting reduced N-type markers growth associated protein (GAP43), synaptophysin (SYP), and tubulin beta 3 (TUBB3) and increased S-type markers Vimentin (VIM), α-smooth muscle actin (α-SMA), and basic calponin (CNN2) mRNA levels in shRNA CgA knockdown cells compared to nonsense control neuroblastoma SH-SY5Y cells. (B–D) qPCR results depicting reduced expression of the glial cell marker (GFAP) (B), but increased expression of Schwannian cell lineage markers, peripheral myelin protein 22 (PMP22) (C) and serpin peptidase inhibitor (SERPINF1) (C), and Schwann cell related extracellular matrix genes, including fibronectin (FN), laminin beta 2 (LAMB2) and type IV collagen (COL4A1) (D) in shRNA CgA knockdown cells compared to nonsense control neuroblastoma SH-SY5Y cells. (E) CgA rescue experiment using an shRNA-resistant CgA plasmid to characterize phenotypic lineage marker changes (SYP for N-type, and VIM and α-SMA for S-type) by real-time PCR. Normalization over nonsense control (A–D) or vector control (E) was used to calculate fold changes. (F) All-trans retinoic acid (atRA)-treatment (20 µM) associated neurite outgrowth was observed in nonsense control neuroblastoma SH-SY5Y cells but not in the shRNA CgA cells treated with atRA. Scale bar: 100 µm, left panel. AtRA-induced cell growth arrest was abolished in shRNA CgA knockdown cells compared to nonsense control neuroblastoma cells (right panel). Proliferation rate fold change was relative luminescence signal to medium control of the nonsense control cells. Each bar indicates the mean±s.d. of triplicate tests. Data were analyzed by two-tailed unpaired t-test with Welch's correction, *P<0.05; **P<0.01; ***P<0.005.
Fig 4: Flank xenografts of neuroblastoma cells lacking CgA show a shift towards an S-phenotype. (A) Comparison of tumor development time in CgA knockdown cells (n=10) and nonsense control neuroblastoma cells (n=10) in an in vivo xenograft model of neuroblastoma. Trend towards a reduction in tumor volumes (B) and weights (C) in the animals bearing CgA knockdown cells compared to nonsense control carrying animals. Note that these results did not attain statistical significance. (D) Representative images of tumor H&E and Vimentin IHC staining (n=4 for each group, left panel, scale bars: 50 mm), percentages of VIM immunoreactive cells (middle panel), and VIM mRNA expression (n=2 for nonsense group, and n=3 for shRNA CgA group, right panel) in two groups. Each bar indicates the mean±standard deviation of triplicate tests. Data were analyzed by two-tailed unpaired t-test with Welch's correction, **P<0.01; ***P<0.005.
Fig 5: CRISPR-Cas9-mediated knockout confirmed the role of CgA in cell proliferation and differentiation. (A) Depiction of the targeting site on CgA Exon 2 chosen for CRISPR/Cas9-directed CgA knockout (top panel), which was confirmed by western blotting (bottom panel). (B,C) CgA sgRNA transfectants exhibited lower proliferation rates compared to control neuroblastoma SH-SY5Y cells measured by CellTiter-Glo® luminescent cell viability assay (B) and BrdU incorporation (C). (D) Quantitative PCR demonstrated that the CgA sgRNA knockout neuroblastoma cells exhibited increased S-type markers (VIM and a-SMA), ECM markers (FN and COL4A1) compared to control neuroblastoma transfectants. Normalization over control cells (B–D) was used to calculate fold changes. Each bar indicates the mean±s.d. of triplicate tests. Data were analyzed by two-tailed unpaired t-test with Welch's correction, *P<0.05; **P<0.01; ***P<0.005.
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