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Figure 5 | Respiratory Research

Figure 5

From: Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells

Figure 5

Susceptibility of A549 cells with ABCA3 mutations to ER stress measured by XBP1 splicing. XBP1 activation during UPR is regulated on the level of splicing of a 26 nt intron from the XBP1 mRNA. (A) XBP1 splicing in untransfected A549 cells with and without tunicamycin (TM) treatment (10 μg/ml, 14 h) and in A549 cells with R43L, R280C and L101P mutations. Two PCR products were detected by RT-PCR: u - unspliced and s - spliced XBP1 bands. h denotes a hybrid band between unspliced and spliced ssDNA produced during PCR and observed after long-run separation on 3% agarose (upper panel) (21). For easier densitometric evaluation and to ensure good separation between unspliced and hybrid band, half of the PCR products were cut by PstI endonuclease which cuts only unspliced (u) band giving two bands u1 and u2 (middle panel). Control reactions without reverse transcriptase (-RT) are shown as well. (C, D) Densitometric quantification of the bands (s, u) in (A) from the middle panel (PstI digest), presented as the ratio s/(u1+u2). TM highly elevated XBP1 splicing in A549 cells. Lower effect of L101P mutation on XBP1 splicing in A549 cells and no effect in A549 with WT and R43L and R280C mutations were observed. (B, E) TM treatment (10 μg/ml, 14 h) strongly induced XBP1 splicing (disappearance of unspliced bands u1 and u2) in all A549 cells expressing ABCA3 mutations, with the most significant increase in A549 expressing R280C and L101P mutations (increase in spliced band s). Shown are (B) 3% agarose gel after PstI digestion of the RT-PCR products (upper panel) and 18S rRNA (lower panel), and (E) densitometric evaluation of the bands. Presented graphs are the densitometric quantification of four independent experiments; * p < 0.05, ** p < 0.01.

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