Stable MAPT-AS1 or miniNAT overexpression does not change Tau expression in SH-SY5Y cells.

a, eGFP expression in SH-SY5Y cells was confirmed after treatment with a SYN1:eGFP construct at a multiplicity of infection (MOI) of 30. Representative images 72 hours after puromycin treatment. Scale bar 400μm. b-i, Expression levels of MAPT (b-d) or MAPT-AS1 (e) and t-NAT (f-i) transcripts in SH-SY5Y cells at passage 3 or 17 post-thawing (#3 or #17 PT, respectively). RNA expression levels were evaluated by RT-qPCR; n = 3 SH-SY5Y lysates per passage; relative RNA level values are normalized to 2 endogenous control genes and calibrated to lowest passage number (#3 PT); data are mean ± SD. j, Tau protein levels in SH-SY5Y cells at passage 3, 11 or 17 post-thawing (#3, #11 or #17 PT, respectively) were assessed using Western Blot analysis; n = 3 SH-SY5Y lysates per passage; Tau protein levels were normalized to β-actin levels and scaled to the lowest passage number (#3 PT; average set to 1); data are mean ± SD. k-s, SH-SY5Y cells were treated with lentiviral constructs at a multiplicity of infection (MOI) of 30 and cells stably expressing eGFP, MAPT-AS1 or miniNAT, with exception for the parental line which remained non-treated. Cell lysates were harvested from 3 consecutive passages for RNA and Tau protein analysis. k-o, Expression levels of miniNAT (k), MAPT-AS1 (l) and t-NAT transcripts (m-o) were evaluated by RT-qPCR; n = 2–3 independent lysates per each condition and per passage; data from different experiments indicated as circles, squares or triangles, respectively; relative RNA level values are normalized to 2 endogenous control genes and calibrated to parental line group; Dunn’s multiple comparisons test (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p < 0.0001). All data are mean ± SD. p, q, MAPT mRNA expression levels in SH-SY5Y stable cell lines were evaluated by RT-qPCR using two independent primer sets; n = 2–3 SH-SY5Y lysates per passage; relative RNA level values are normalized to 2 endogenous control genes and calibrated to parental line group; Dunn’s multiple comparisons test. r, s, Tau protein levels from SH-SY5Y lines were assessed using a full-length Tau protein MSD assay and normalized to total protein (r) or Western Blot analysis and normalized to β-actin levels (s); n = 2–3 independent lysates per each condition and per passage; values scaled to parental line group; One-way ANOVA with Holm-Šídák’s multiple comparisons test (*, p ≤ 0.05).

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