Fig 2: The presence of JAB1 is required for AP1-driven transcription in TNBC cells.a BT549 or MDA-MB-231 cells were treated with 30 nM JAB1 siRNAs (siJAB1-1, siJAB1-2) or scrambled siRNA control for 3 days followed by western blot analysis to detect JAB1, cyclin D1, uPA, uPAR, and GAPDH with the respective antibodies. Data are the representative of two independent experiments. All blots derived from the same experiment and were processed in parallel. b BT549 or MDA-MB-231 cells were treated with 30 nM JAB1 siRNAs or scrambled siRNA control for 3 days followed by qRT-PCR to measure the levels of Cyclin D1, uPA and uPAR mRNA. Level of βActin mRNA was used for standardization. Data are means ± SD from three experiments. *P < 0.01 vs Control; #P < 0.005 vs Control. c, d BT549 or MDA-MB-231 cells were treated with 30 nM JAB1 siRNAs or scrambled siRNA control for 3 days and then subjected to RP-II (c) or JUN ChIP (d) followed by qPCR to analyze RP-II or JUN occupancy in cyclin D1, uPA and uPAR promoters. Data are means ± SD from three experiments. * P < 0.01 vs Control.

Fig 3: The presence of JAB1 is critical for tumor development.a Luciferase-expressing MDA-MB-231 cells were transduced with lentiviral vector encoding either scramble or JAB1 shRNA for 3 days and then subcutaneously injected into nude mice. Tumor outgrowth was monitored weekly using the Xenogen IVIS-200 In Vivo bioluminescence imaging system. Data are means ± SD. n = 5. Statistical analysis was performed using Student t test (two-tailed) for the end experiment point. *P < 0.001. b Images of the xenograft tumors at 5 week. The image data is displayed in radiance or photons/sec/cm2/steradian. c Luciferase-expressing MDA-MB-231 cells were injected into nude mice for 5 days. Mice were randomized and treated daily with either Vehicle control or 1 µmol/kg of SB203580 i.p. for 4 weeks. Tumor growth were monitored weekly though Xenogen IVIS-200 in vivo imaging system. Data are means ± SD. n = 5. Statistical analysis was performed using Student’s t test (two-tailed) for the end experiment point. #P < 0.05. d Images of the xenograft tumors before treatment and after 4 weeks of treatment.

Fig 4: JAB1 is an MK2 substrate and is required for robust AP1 activity in TNBC cells.a MDA-MB-231 cells were immunoprecipitated with either JAB1 or MK2 antibody and the immunoprecipitates were subjected to western blot to detect MK2 or JAB1 respectively. All blots derived from the same experiment and were processed in parallel. b MDA-MB-231 cells were subjected to immunofluorescence staining to detect JAB1 and MK2. Data are the representative of four independent experiments. c Diagram of HA-tagged full-length JAB1, N-terminal Jab1 (JAB1-N), and C-terminal JAB1 (JAB1-C). d HEK293 cells were transfected with empty plasmid (Control) or with plasmids encoding HA-tagged full-length JAB1 (FL), JAB1-N or JAB1-C, and cell lysates were immunoprecipitated with MK2 pAb. Immunoprecipitates were subjected to western blot analysis to detect interactions between the JAB1 proteins and MK2 by HA mAb. Data are the representative of two independent experiments. All blots derive from the same experiment and were processed in parallel. e BT549, MDA-MB-231 or MDA-MB-436 cells were treated with 30 nM JAB1 siRNA (siJAB1-1 and siJAB1-2) or scrambled siRNA control for 3 days followed by analyzing AP 1 activity. Data are means ± SD from three experiments. *P < 0.01 vs Control. f Wild-type MEFs were first transduced with MKK3D, MKK6D, MK2-CA, or empty vector (Control) for 3 days and then treated with 30 nM JAB1 siRNAs for 3 days followed by analyzing AP1 activity. Data are means ± SD from three experiments. *P < 0.01 vs Control.

Fig 5: Ser177 phosphorylation of JAB1 impacts JUN binding to AP1 sites.a BT549 or MDA-MB-231 cells were treated with 5 µM SB203580 for 1 day or 30 nM MK2 siRNA (siMK2-1) for 3 days for left untreated (control) and then subjected to JUN ChIP followed by qPCR to analyze JUN occupancy in cyclin D1, uPA and uPAR promoters. Data are means ± SD from three experiments. *P < 0.01 vs Control. b BT549 or MDA-MB-231 cells were transduced with JAB1D, JAB1A, or empty vector (control) for 3 days and then subjected to JUN ChIP followed by qPCR to analyze JUN occupancy in cyclin D1, uPA and uPAR promoters. Data are means ± SD from three experiments. *P < 0.01 vs Control. c, d BT549 (c) or MDA-MB-231 cells (d) were transduced JAB1D or empty vector for 3 days and then treated with 5 µM SB203580 for 1 day or 30 nM MK2 siRNA (siMK2-1) for 3 days followed by JUN ChIP to detect JUN occupancy in cyclin D1, uPA and uPAR promoters. Data are means ± SD from three experiments. *P < 0.01 vs Control.