transient transfection hek293t  (ATCC)

Journal: Nature communications

Article Title: PIEZO1 loss-of-function compound heterozygous mutations in the rare congenital human disorder Prune Belly Syndrome.

doi: 10.1038/s41467-023-44594-0

Figure Lengend Snippet: Fig. 3 | Evaluation for loss-of-function effect of Piezo1 PBS mutations in HEK293TΔP1 cells. a Representative confocal images (n = 12) of HEK293TΔP1 cells co- expressing WT-tdTom with either S260R-GFP (upper) or S2211L-GFP (lower) fused proteins. b Single-channel current recordings of WT and PBS variant proteins expressing WT-tdTom alone, WT-tdTom with either S260R-GFP or S2211L-GFP. WT- GFP with S2211L-GFP was used as control. c 30 s all-point current histograms of the single-channel recordings shown in (b). d Steady state NPo of WT-dtTom and PBS- GFP variants. Statistical analysis was performed by using One-way ANOVA where ***p < 0.001, ****p < 0.0001 and ns = not significant. tdTom = tdTomato, C = close, O = open, pA = picoamperes, NC = normalized counts, NPo = normalized open probability, calibration bar = 10 µm. Data points in (d) are represented as Mean ± SD.

Article Snippet: Primers for sequencing were purchased from IDT and are as follows: S260R (forward):5’-CTGGTGGTCCTGTCACTTTC-3’ S260R (reverse):5’-GCTCTGGCTGGTTAGTACAT-3’ S2211L (forward):5’-AGCCGAGAGACAGAGAAGAA-3’ S2211L (reverse):5’-CTCAGGACTGTACTGGCTAATG-3’ C-term w/2422-Myc tag (forward): 5’-TTCCCCATCTCTTCCCC AAG-3’ C-term w/2422-Myc tag (reverse): 5’- GGAAGATGAGCTTGGC GTATAG −3’ Cell culture and transient transfection HEK293TΔP1 cells (PIEZO1 Knockout cell line) were purchased from ATCC (#CRL-3519).

Techniques: Expressing, Variant Assay, Control

Journal: American journal of medical genetics. Part A

Article Title: ALDH1A2 -related disorder: A new genetic syndrome due to alteration of the retinoic acid pathway

doi: 10.1002/ajmg.a.62991

Figure Lengend Snippet: Biallelic ALDH1A2 sequence variants segregate with phenotype and alter expression levels of the gene product. (a). Pedigree showing segregation of developmental syndrome with biallelic ALDH1A2 sequence variants. (b). The indicated variants were introduced into a mammalian expression construct encoding HA-ALDH1A2 and transfected into HEK293T cells. Western blot analysis reveals reduced expression of the p.(Arg347His) variant and absent expression of p.(H253Qfs * 4). (c). Relative protein expression was quantitated in five independent experiments, ****p < 0.0001. (d). Immunofluorescence assay showing reduced expression of the p.(Arg347His) variant. (e). Semi-quantification for immunofluorescence intensity was measured in 5 cells per frame for a total of 25 cells. Corrected total cell fluorescence (CTCF), **p < 0.01

Article Snippet: Cell culture and transient transfection assays HEK293T cells were from American Type Culture Collection (CRL-11268), were maintained in high glucose DMEM media (11995065) supplemented with 10% FBS and Penicillin-Streptomycin, and grown in a 5% CO2 humidified incubator at 37°C.

Techniques: Sequencing, Expressing, Construct, Transfection, Western Blot, Variant Assay, Immunofluorescence, Fluorescence