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. 2015 Nov 20;11(11):e1005274. doi: 10.1371/journal.ppat.1005274

Fig 12. Depletion of Hsc70 abrogated KSHV RTC formation in TREx BCBL1-RTA cells.

Fig 12

(A) Cells were nucleofected with either scramble or Hsc70-specific siRNA together with pmaxGFP to monitor transfection efficiency. 48 h post-nucleofection cells were imaged by fluorescent and light microscopy with a 40-times objective. Merge images are shown. (B) After four days post-nucleofection, total RNA was isolated and qRT-PCR carried out. Hsc70 mRNA levels showed ~ 90% knockdown, in contrast iHsp70 mRNA was significantly increased. (C) A minor Hsc70 depletion at the protein level was observed at four days post-nucleofection while iHsp70 was clearly upregulated in Hsc70 siRNA-treated cells as shown by Western blotting. (D and E) Six days post-nucleofection cells were reactivated for 24 h and immunofluorescence was performed with RTA and Hsc70 specific antibodies. (D) In scramble siRNA-treated cells RTCs to which Hsc70 was recruited were observed. (E) In Hsc70 siRNA-treated cells, cells nearly or completely depleted of Hsc70 protein did not form RTCs (white arrows), while cells presenting assembled RTCs still displayed Hsc70 nuclear foci (yellow arrows).