Presentation on theme: "VectorRNAimismatch 12-14-16 c 16-18-20 c linearity EGFP SERCA1b vectorRNAimismatch ** + SI 1 SI 1: Cotransfection of the SERCA1b RNAi and the SERCA1b expressing."— Presentation transcript:
vectorRNAimismatch c c linearity EGFP SERCA1b vectorRNAimismatch ** + SI 1 SI 1: Cotransfection of the SERCA1b RNAi and the SERCA1b expressing plasmid into COS- 1 cells decreased the SERCA1b mRNA level. The PCR amplifications were made in the linear range for both SERCA1b (18 cycles) and EGFP (14 cycles) as depicted in the inset. ** p<0.01 with respect to vector, + p<0.05 with respect to mismatch control.
DC A B SI 2
SI 2: Controls for GFP expression in transfected fibers. Green fluorescence image (AC) and anti-GFP staining (BD) of paralel sections of GFP-transfected (AB) and nontransfected (CD) 7 days regenerating soleus muscles. Arrows on AB point to fibers positive for both GFP and GFP-immunostaining. Note that not all GFP positive fibers are detected by immunohistochemistry. The exposure time for the nontransfected soleus on C was twice as long as for the GFP-transfected muscle on A. Bar on D 100 m.
RED GFPOverlay A B REDGFP SI 3
SI 3: Cotransfection and successive transfection. Fibers transfected with pEGFP (GFP) and/or pDsRED (RED) are also shown in overlay. Note that (a) after cotransfection the same fibers express EGFP and RED while (b) after successive transfection different fibers express EGFP and RED. Bar 100 m.
SI 3. 11d V RNAi RNAi + cain RNAi //cain 28dN SI 4 SI 4: The phenotype of the transfected regenerating muscles. 11d and 28d – nontransfected muscles regenerated for different lenghts of time indicated as number of days. 11-days regenerating soleus muscles transfected with empty vector of RNAi (V) and SERCA1b RNAi (RNAi). 11-days-regenerating soleus muscle cotransfected (RNAi + cain) or separately transfected (RNAi//cain) with SERCA1b RNAi and cain. N – normal soleus of rat of the same age (four-months).
SI 5 SI 5: Visualization of myonuclei. The immunostaining of sarcolemma for - sarcoglycan and the nuclear staining with DAPI. Bar 50 m.