Figure 1

Activated JAK-STAT signaling pathway in MV human diaphragm. (A) Altered signaling pathways in the MV human diaphragm. Altered genes (p < 0.05) from microarray analysis of MV versus control diaphragm samples were input into DAVID Bioinformatics analysis. In the pie figure, the percentage indicates the number of genes involved in the specific pathway over the total number of genes involved in all signaling pathways listed. The p value indicates the significance of the altered signaling pathway. Signal pathways with p < 0.05 are listed. (B) The mean fold change in MV versus control human diaphragm samples of genes significantly (p < 0.05) altered by MV that are involved in the JAK-STAT signaling pathway (n = 4 pairs of samples). The color green in the heat map indicates the reduced expression of genes, while red indicates increased expression. Specific fold changes are indicated in the right column. (C) Quantitative PCR was performed to confirm the altered expression of genes identified by microarray. Human diaphragm muscle samples (Con n = 6, MV n = 8) were examined, *p < 0.05. (D) Activation of the JAK-STAT signaling pathway. Protein extracts from MV and control human diaphragm muscles were subjected to Western blot analysis (n ≥ 4). The two lower bar graphs show the data quantified, with normalization of phosphorylated protein. *p < 0.05. (E) Nuclear enrichment of STAT3 in MV human diaphragm. Human diaphragm muscles were stained with STAT3 (red), DAPI (blue) or WGA (green) to show STAT3, nuclei and cell membrane respectively. Arrows indicate the location of nuclei. The lower panel shows the quantitated data of the STAT3-positive nuclei per total nuclei. n = 4, *p < 0.05. Note that STAT3 is enriched in nuclei in the ventilated but not the control human diaphragms. (F) STAT3 is not activated in nonrespiratory muscles during mechanical ventilation. Protein extracts from human quadriceps muscles from control and MV patients, and subjected to Western blot analysis. Note that there is no induction of phosphorylated STAT3 in quadriceps muscles under MV F.C., fold changes; Con, control; IL6R, interleukin-6 receptor; IL20Ra, interleukin-20 receptor alpha subunit; IL20Rb, interleukin-20 receptor beta subunit; a.u., arbitrary unit.