Fig 2: TPR co-immunoprecipitates with LSD1 in C2C12 MBs and targets it to Myh4. (a) Immunoprecipitation of TPR by anti-LSD1 (top), immunoprecipitation of LSD1 by anti-TPR-C (bottom) in C2C12 MBs. IgG served as a negative control. (b) ChIP-qPCR analysis of LSD1 binding to Myh4 n (WT, shTPR1) = 4, n (shTPR2) = 2; Mef2C n (WT, shTPR1) = 3, n (shTPR2) = 2, Olfr376 n (WT, shTPR1) = 4, (shTPR2) = 3; and Myog n (WT, shTPR1) = 3, n (shTPR2) = 2. Data normalized to H3 binding in respective sample; H3 binding is equal to 1 = threshold. Data were square root transformed prior to statistical evaluation using Welch’s t-test. Each dot represents one biological replicate. (c–e) Protein levels of LSD1, TPR and MYH4 in C2C12 control (WT, or transfected by non-targeting siRNA; siNC) and C2C12 cells depleted of LSD1 (siLsd1) or TPR (siTpr) using esiRNAs. Each dot represents one biological replicate, n (WT, siNC, siLSD1) = 6, n (siTPR) = 5. (f) Phase contrast images of MTs (differentiated for 4 days) arising from WT, siNC, siLsd1 and siTpr depleted MBs. Error bars represent s.e.m.; p < 0.01; *, p < 0.05; ns, non-significant.

Fig 3: TPR is important for C2C12 myogenic differentiation. (a) Growth curve of WT and TPR-depleted C2C12 cells shows decreased proliferation rate (see proliferation phase, P) in TPR-depleted C2C12 cells. WT cells started to differentiate at a confluency of 106/cm2. TPR-depleted cells continued to proliferate up to an almost tripled confluency compared to the WT (arrows, differentiation phase, D). The cells were counted until the second day of differentiation (gray dashed line, MT2. The graph represents one biological replicate. (b) Phase contrast images of WT and TPR-depleted fully confluent MT0 and MTs (differentiated for 0 and 4 days, respectively). (c,d) Dot plots show decreased MT width and fusion index in TPR-depleted C2C12 cells in comparison to WT. Each dot represents one biological replicate out of three. Data were square root transformed for statistical evaluation: Welch’s t-test, n (each cell line) = 100 cells, one dot represents one cell. (e–g) TPR depletion did not affect Myog, (n (WT, shTPR1) = 6, n (shTPR2) = 5) and Mef2C mRNA levels in MTs (n = 5), but resulted in a reduction of Myh4 mRNA levels in MTs (n = 5). (h–j) No difference was observed for MYOG (n = 6) and MEF2C protein levels in MTs (n = 4); the decreased expression was confirmed for MYH4 protein levels in MTs (n = 6). Each dot represents one biological replicate. qPCR and WB data were log-transformed prior to statistical evaluation: Student’s one sample t-test was used to test against the normalized WT MB value set to 1; Welch’s t-test was used for between-sample comparisons. Error bars represent s.e.m.; *, p < 0.05; ***, p < 0.001.; ns, non-significant.

Fig 4: PECAM1 and CXCR4 are highly expressed in the inflamed dental pulp tissues. (A) Aberrantly activated signaling pathways in the inflammatory dental pulp tissues predicted by GSVA. (B–C) The interacted proteins with PEACM1 and the highly correlated genes predicted by PPI analysis. (D–E) Enrichment of signaling pathways based on the differentially expressed genes predicted by GSEA. (F) Concentration of 22 types of immune cells in each sample determined using an R/Cibersort Package. (G–H) Predicted binding sites between MEF2C with CXCR4 and PECAM1.

Fig 5: Validation of causality between differentially expressed genes and 22q11.2del in an isogenic setting.a Generation of isogenic lines with 22q11.2del using CRISPR/Cas9 guide RNAs that cut within the low copy repeats (LCRs) flanking the 3 Mb deletion. Coordinates for the guides genomic position on chromosome 22 are indicated (Hg19). b Detection of isogenic 22q11.2del using DNA FISH analysis and a probe generated probe using CTD-2300P14 (Thermo Fisher Scientific, 96012). Blue = DAPI (DNA), Red=22q11.2 region. Scale bar: 10um. (N(edited clones) = 2 and N(nonedited clones) = 2; 3 experimental replicates each). c ddPCR assay to determine the copy numbers of HIRA and ZNF74, located in 22q11.2 (N=2 wildtype and 2 edited clones and 1 patient control). Analysis performed via QuantaSoft software (BioRad); copy number for HIRA and ZNF74 (normalized to RRP30), error bars represent the Poisson 95% confidence limits. d SNP array marker intensity (LRR) for SNPs overlapping the deletion locus confirms 22q11.2del in two clones (red). e,f Principal component analysis of cell lines with and without isogenic 22q11.2del. Circles = genes within 22q11.2 (cis). Triangles = genes outside 22q11.2 (trans). e PC1 and PC2 separate cells by developmental stage., PC3 and PC4 separate cells by deletion genotype. g Significant downregulation of genes in 22q11.2 in lines with isogenic 22q11.2del (Mann-Whitney U test for 32 genes in 2 deletion and 2 control clones, two-sided). Data is presented in a Tukey-style boxplot with the median (Q2) and the first and the second quartiles (Q2, Q3) and error bars defined by the last data point within +/- 1.5-times the interquartile range. h Correlation of fold changes in differentially expressed genes in discovery and isogenic datasets in neurons. Transcripts from 32 genes were detected and significantly changed in the discovery and isogenic lines (adjusted p-value < 0.05), of which nine were located outside 22q11.2 (FAM13B, KMT2C, HYAL2, DNPH1, ZMYM2, VAPB, SMG1, CPSF4, MAP3K2). All 32 genes were changed in the same direction in both cohorts (p = 5.6 × 10-9, binomial test). Genes with a SynGO annotation shown in red, genes with no SynGO annotation shown in blue. Circles = cis genes. Triangles = trans genes. i, MEF2C is upregulated in 22q11.2del NPCs compared to isogenic controls (N(edited clones) = 2 and N(non-edited clones) = 2; 3 experimental replicates each). Data is presented in a Tukey style boxplot with the median (Q2) and the first and the second quartiles (Q2, Q3) and error bars defined by the last data point within +/- 1.5-times the interquartile range. j SynGo annotation of genes induced in isogenic 22q11.2del neurons showing enrichment for synaptic vesicle cycle and endocytosis.