Expression of CLMP in T84 cells transduced with wild-type (WT)-CLMP, mutant-CLMP (V124D), RFP and an empty vector.

<p>There is no endogenous expression of CLMP in T84 cells (see the right lane (empty vector)). A. WT-CLMP and mutant-CLMP (V124D) mRNA were equally expressed in the transduced T84 cells as measured by real-time PCR. B. Western blots showed that WT-CLMP and mutant-CLMP (V124D) (at 41 kDa) protein were equally expressed. The 100 kDa band is an aspecific band derived from the vector.</p

No significant difference was observed in cell aggregation between CHO cells transfected with and without CLMP (wild-type and mutant (V124D)).

<p>A. Mock, CHO cells that were not transfected. B. CHO cells transfected with CDH1. C. CHO cells transfected with wild-type-CLMP. D. CHO cells transfected with mutant-CLMP (V124D).</p

Overexpression of wild type (WT)-CLMP and mutant-CLMP (V124D) in human intestinal epithelial T84 cells does not affect wound healing/migration, cell proliferation, viability, and trans-epithelial electrical resistance.

<p>A. Cell monolayers were wounded and incubated in serum-deprived medium for 24 hours. The rate of directional cell migration (distance travelled/time unit) and wound closure were determined. B. Proliferation was quantified measuring BrdU incorporation using a BrdU cell proliferation assay. There was no significant difference in the specific optical density (OD) (the measured OD at wavelength 370 nm minus the measured OD at wavelength 492 nm) between the three groups. C. Cell viability was estimated using an <i>in vitro</i> Toxicology Assay Kit XTT-based. XTT was added to the cells at a final concentration of 20 µg/ml. After 4 hours of incubation the OD was measured at 450 nm using the 690 nm absorbance as the background. There was no significant difference between the three groups. D. Transepithelial electrical resistance was measured in confluent cell monolayers cultured on Transwell filter inserts.</p

Additional file 9: Table S8. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Characteristics of 116 ENS-related HSCR candidate genes. (XLSX 32 kb

Additional file 7: Table S6. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Gene recurrence and burden test. (XLSX 14 kb