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. 2015 Apr 2;2015:645157. doi: 10.1155/2015/645157

Figure 4.

Figure 4

Impairment of Hsp70 prevents upregulation of BiP but not of CHOP. (a) Western blot was performed with anti-BiP or anti-CHOP followed by peroxidase-conjugated specific secondary antibody to analyze these proteins in the lysates of untreated cells or Q (10 μM) or 4μ8c (10 μM) pretreated cells and thereafter treated with none or TN for 24 h. Western blot of Hsp90 is shown at the bottom as a loading control. Representative blots are shown. Densitometric quantification of the bands is shown at the bottom of the relevant lines as the ratio of BiP or CHOP in each line, respectively, to the BiP or CHOP values observed in the lysate of untreated U937 cells or to the CHOP value observed in TN treated cells. Western blot analysis of Hsp90 is shown at the bottom, as a loading control. (b) Western blot analysis of BiP or CHOP by specific antibody in the lysates of U937 cells transfected with equal amounts of Hsp70 siRNA or scr-siRNA (Santa Cruz) or equal amounts of IRE1α siRNA or scr-siRNA (Ambion) for 72 h and exposed to none or to TN 1 μM for further 24 h. Western blot analysis of β-actin is shown at the bottom, as a loading control and also to confirm the specificity of the transfected siRNA. Representative blots are shown. Densitometric quantification of the bands is shown at the bottom of the relevant lines as the ratio of BiP or CHOP in each line, respectively, to the BiP or CHOP values observed in the lysate of untreated U937 cells transfected with scrambled siRNA (Santa Cruz). (c) Detection of cell death by evaluation of sub-G1 events. U937 cells were pretreated for 30 min with none or 4μ8c (10 μM) and thereafter for 24 h with none or TN (1 μM). The cells were fixed and stained with PI to evaluate sub-G1 events in the cell cycle by cytofluorimetric analysis performed on ≥10000 events. The values reported are means ± S.D. (n = 5). Assessment of cell death showed statistically significant differences between the data obtained in the cultures treated with TN and 4μ8c in comparison with the cultures treated only with TN (∗ P < 0.0001).