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. 2022 Apr 7;20:47. doi: 10.1186/s12964-022-00855-x

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — NOS2 mRNA isoform analysis using 5′RACE and isoform specific qRT-PCR methods. A Schematic summary of the results of 5′-RACE analyses using RNA isolated at day 18 from human iPSC induced to differentiate to neurons. RNA was isolated from 4 different human iPSC lines (samples C16-1, C16-2, C89-2, C89-3) induced to differentiate to neurons [53] for 18 days. As control RNA was isolated from human DLD1 cells (DLD1-2 and DLD1-2) treated with a cytokine mixture for 6 h to induce NOS2 mRNA expression. Then 5′-RACE experiments were performed (as described in “Methods” section) using the indicated oligonucleotides as 3′-primer (5′RACE-rev = primer used for the RT-reaction; 3P2/3P3 primers used for the PCR-reaction). The final PCR products were isolated, cloned into pCR-Script and sequenced. The alignment of these sequences to the human NOS2-1 and -2 mRNA are indicated as filled arrows. The sequences of the fragments are shown in Additional file 1: Fig. S10. B/C NOS2-mRNA-isoform expression analysis using isoform specific qRT-PCR experiments. RNA was isolated at different time points from 2 different human iPSC lines (iLB-C16bm s6 iPS andiLB-C89bf s4 iPCs) induced to differentiate to neurons [53] for 60 days. The RT reaction was performed with the RT-rev primer (see A). Taqman qPCR reactions were performed with mRNA isoform specific primer pairs and taqman probes (NOS2-1: NOS2-1_P, NOS2-1_3P and NOS2-1_Taq; NOS2-2: NOS2-2_5P, NOS2-2_3P and NOS2-2_Taq—see A). For normalization also the GAPDH mRNA expression was measured. B The normalized NOS2-1 mRNA expression values were related to the CM induced NOS2-1 mRNA expression in DLD-1 cells (CM = 100%). Also, the expression level of the NOS2-1 mRNA in untreated (co) DLD-1 cells were determined. (***p < 0.001, ns not significant vs. iPSC treated for 18 days; 1-way Anova with Dunnett’s multiple comparisons test). C The normalized NOS2-2 mRNA expression values were related to the NOS2-2 mRNA expression in iPSC treated for 18 days (day 18 = 100%). (**p < 0.05, vs. iPSC treated for 18 days; 1-way Anova with Dunnett’s multiple comparisons test)