![Figure 1. DGK-α is required for α5β1 recycling in response to αvβ3 inhibition or expression of mutant p53. (A–E) The recycling of internalized α5β1 (A–D) or 125I-transferrin (TFN; E) was determined in A2780 (A–C and E) or empty vector and mutant p53-expressing H1299 (D) cells. In A and E, A2780 cells were transfected with a nontargeting siRNA (siRNA control [si-con]) or an siRNA targeting DGK-α (si-DGKα). In C, A2780 cells were transfected with an siRNA targeting DGK-α or siRNA targeting DGK-α in combination with an siRNA-resistant DGK-α (si-DGKα/DGKα(resc)) or empty vector control (pMT2). (B and D) The DGK inhibitor, 1 µM R59949, or DMSO control was included during the internalization and recycling periods as indicated. (E) 120 µM chloroquine (CQ) was included during the internalization and recycling periods, whereas 0.3 mM primaquine (PQ) was only in the recycling period. (A–C) 36 nM cRGDfNMeV (A and B) or 2.5 µM cRGDfV (C) was included as indicated during the recycling period. Values are means ± SEM (A–D) and means ± SD (E) of at least nine replicates. †††, P < 0.0001 for si-con+cRGDfNMeV versus si-DGK-α+cRGDfNMeV; Mann–Whitney test. ***, P < 0.0001 for si-DGK-α+cRGDfNV versus si-DGK-α/DGK-α(resc)+cRGDfV; Mann–Whitney test. §§, P < 0.0001 for DMSO+cRGDfNMeV versus R59949+cRGDfNMeV; Mann–Whitney test. ‡‡‡, P < 0.0001 for p53273H DMSO versus p53273H R59949; Mann–Whitney test. $$$, P = 0.0002 for vehicle versus primaquine/chloroquine; Mann–Whitney test.](https://cdn.rupress.org/rup/content_public/journal/jcb/196/2/10.1083_jcb.201109112/6/jcb_201109112_fig1.jpeg?Expires=1771704456&Signature=MM1fBubktwgn8R-sHNJkVvDHCsKNFn8LirfoQHndLIEQ6VUMdKzKPEHQAEFOmWOT8T1wmhzkThDWG9pojhf5LpYzNngMQMd4FtYQJNbsXOSCG~7sXqeykm6vkui5wJVvSrto4QSja7ofLNv-XSCMRUj1j-RJvNvJ8pMknPTU1lVlk1Kra6SZH6nHVcfMbUbh1StoLy~xYsQdLgbhU4uDQ1JsWgX0bbo0JdsuLbK90zhM8mMriQmHsiVqTOE1cgl2TtU1Tf8EHM9hDY9j7lvnLMOY03X1IjtlHYPHxb0Ux48HQ7t0PEdZu1TgvAnRpQKnuOAa24Z9uBQEMxeTfUjZUw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
DGK-α is required for α5β1 recycling in response to αvβ3 inhibition or expression of mutant p53. (A–E) The recycling of internalized α5β1 (A–D) or 125I-transferrin (TFN; E) was determined in A2780 (A–C and E) or empty vector and mutant p53-expressing H1299 (D) cells. In A and E, A2780 cells were transfected with a nontargeting siRNA (siRNA control [si-con]) or an siRNA targeting DGK-α (si-DGKα). In C, A2780 cells were transfected with an siRNA targeting DGK-α or siRNA targeting DGK-α in combination with an siRNA-resistant DGK-α (si-DGKα/DGKα(resc)) or empty vector control (pMT2). (B and D) The DGK inhibitor, 1 µM R59949, or DMSO control was included during the internalization and recycling periods as indicated. (E) 120 µM chloroquine (CQ) was included during the internalization and recycling periods, whereas 0.3 mM primaquine (PQ) was only in the recycling period. (A–C) 36 nM cRGDfNMeV (A and B) or 2.5 µM cRGDfV (C) was included as indicated during the recycling period. Values are means ± SEM (A–D) and means ± SD (E) of at least nine replicates. †††, P < 0.0001 for si-con+cRGDfNMeV versus si-DGK-α+cRGDfNMeV; Mann–Whitney test. ***, P < 0.0001 for si-DGK-α+cRGDfNV versus si-DGK-α/DGK-α(resc)+cRGDfV; Mann–Whitney test. §§, P < 0.0001 for DMSO+cRGDfNMeV versus R59949+cRGDfNMeV; Mann–Whitney test. ‡‡‡, P < 0.0001 for p53273H DMSO versus p53273H R59949; Mann–Whitney test. $$$, P = 0.0002 for vehicle versus primaquine/chloroquine; Mann–Whitney test.
