Table 1. Evaluation of antisense activity after co-injection of 30 µM P=S ODNs and expression vectors into the same nucleus of Ref52 cells (A) and evaluation of antisense activity of shuttling P=S ODNs (B).

Sample	Injected	Positive	Percent
(A) Co-injections into the same nucleus
12182-T + TM1	74	4	5
	63	13	21
12182-T + TM4	117	101	86
	70	62	89
10366-X + TM1	74	59	80
	86	66	77
(B) Consecutive injections into different nuclei of binucleate cells
12182-T/TM1	25	1	4
	10	1	10
12182-T/TM4	11	9	82
10366-X/TM1	32	30	94
	18	13	72

First P=S ODNs were injected at 60 µM into one nucleus of binucleate Ref52 cells. Two hours later the respective expression vector was injected into the other nucleus that did not receive the P=S ODN. P=S ODN 12182–Texas Red was complementary to sequences in exon 5 of tropomyosin isoform TM-1 but not TM-4. P=S ODN 10366–XRITC was a control P=S ODN without complementary sequences in TM-1. Sites of injections were verified by 70 kDa dextran–Cascade Blue (site of P=S ODN injection) and fluorescein (site of expression vector injection) co-injections. Expression of the tropomyosin isoforms was checked 3 h after vector injection by indirect immunofluorescence with an antibody against the HA tag of the expressed protein and Cy5-conjugated secondary antibodies. The results of independent experiments are shown where the number of successfully injected cells were scored positive or negative for tropomyosin expression and the percentage of expressing cells calculated.