File S1 - Functional Impacts of NRXN1 Knockdown on Neurodevelopment in Stem Cell Models

Figure S1. Quantitative real-time PCR (a) and Western blot (b) show that HEK-293T cells have native NRXN1 expression including α-NRXN1 and two short β-NRXN1 isoforms. S1, S2, S3, S4: Four HEK-293T cells from 4 different frozen stocks. Figure S2. Evaluation of the knockdown efficiency of shRNAmir clones against NRXN1 using HEK-293T cells. All three shRNAmir clones have significant knockdown of α-NRXN1 shown by quantitative real-time PCR (A) and Western blot (B). sh1: shRNAmir clone V2THS_246980; sh2: shRNAmir clone V2THS_68983; sh3: shRNAmir clone V2THS_246996. Each shRNAmir clone has 4 biological replicates for knockdown experiments in the HEK-293T cells, labeled as 1,2,3,4. w, abbr. of week. Figure S3. Quantitative real-time PCR (a) and Western blot (b) show that 5 days after Doxycycline induction in H9-derived NSCs, three shRNAmir have different but significant knockdown efficiency. sh1: shRNAmir clone V2THS_246980; sh2: shRNAmir clone V2THS_68983; sh3: shRNAmir clone V2THS_246996. Figure S4. Doxycycline induced highly expressed RFP reporter gene inside neurons (A) and astrocytes-like cells (B) in NSCs derived from H9 or iPS at week 2, week 3 and week 4 post differentiation. Figure S5. RNA-Seq data indicated that the shRNAmir (V2THS_68983) also has significant knockdown efficiency to two major β-NRXN1 isoforms on RefSeq ID NM_004801.4 (A) and NM_138735.2 (B) on week 4 of hiPSCs. w, abbr. of week. Figure S6. Dynamic gene expression along entire development and adulthood in the cerebellar cortex (CBC), mediodorsal nucleus of the thalamus (MD), striatum (STR), amygdala (AMY), hippocampus (HIP) and 11 areas of neocortex (NCX), in the developing human brains. Week 4 in our study appears to correspond to period 4 (13–16 post-conceptional weeks) in the Human Brain Transcriptome data generated by Kang et al (2011). Table S1. A list of genes with significantly altered gene expression levels at week 4 as a result of NRXN1 knockdown by shRNAmir (FDRTable 1, based on the UniProt Knowledge Base. (DOC)