Fig. 4: Subcellular localisation and knockdown of circRNAs in vitro.
From: Circular RNAs regulate neuron size and migration of midbrain dopamine neurons during development
a Single-molecule fluorescence in situ hybridisation (smFISH) of circFat3, circLmx1a, circRmst and a scrambled control (Scr) in E14 vMB primary cultures at DIV5. In merged image: DAPI = blue, TH = white, smFISH probe = red. White arrows indicate signal in mDA neurons. Scale bars = 20 µm. b–e Quantification of smFISH in vMB cultures as in a. b Probe signal in mDA neurons in vMB primary cultures at DIV1 versus DIV5. Data presented as percentage of positive mDA neurons (of all mDA neurons). c Quantification of detected signal (punctae) per cell (punctae per DAPI signal) at DIV1 versus DIV5. d Quantification of detected signal (punctae) per mDA neuron at DIV1 versus DIV5. e Quantification of subcellular localisation (soma vs. neurites) of circRNAs at DIV5. b–d n = 3 cultures (DIV1), n = 4 cultures (DIV5), (e) n = 3–4 cultures. Data are shown as means ± SD, (b–d) multiple unpaired two-sided t-tests with Holm Sidak multiple testing, (e) ordinary one way ANOVA with Tukey’s multiple comparisons test, (b–e) all n.s. adjusted P > 0.05. f Schematic overview of the selection of circRNA candidates for KD experiments. g Schematic representation of shRNA design. Three shRNAs were designed for each BSJ. One directly on top of the BSJ (design #1, ‘shRNAI’), 3 nts upstream (design #2, ‘shRNAII’) and 3 nts downstream (design #3, ‘shRNAIII’). h Schematic representation of circRNA knockdown (KD) in primary cultures of dissociated E14 WT vMBs. Corresponding shRNAs were delivered via LV infection to control and KD cultures, respectively. At DIV3, circRNA KD cultures were subjected to RT-qPCR for KD validation. Figure contains icons from BioRender.com. circRNA KD validation as relative expression of circRNA or linear counterpart RNA to control cultures for (i) circEzh2, (j) circRmst and (k) circTulp4. For each circRNA candidate three shRNAs were designed. Results of all constructs with successful plasmid cloning are shown. Sh-circEzh_I, sh-circRmst_I and sh-circTulp_II were selected for further experiments. n = 3 primary cultures, multiple unpaired two-sided t-tests, with Holm-Sidak multiple testing, mean ± SEM, (i) **P = 0.007175, (j) ***P = 0.000694, ****P = 0.000053, (k) ****P = 0.000001. Source data are provided as a Source Data file.
