Fig 2: Rap1 activation mitigates the epithelial barrier dysfunction caused by RSV infection. 16 HBE cells were infected with RSV or control medium for 22 h and DMSO (vehicle) or 5 µM 8-pCPT-AM was added for another 2 h. Treatment of 8-pCPT-AM alleviated reduced resistance and increased permeability caused by RSV. (A) TEER (O cm2) was measured with a volt-ohm meter and plotted as percentage versus control+vehicle. Data shown as mean±s.e.m., n=8 replicates, one-way ANOVA followed by Tukey's multiple comparisons test. Control+vehicle versus control+5 µM 8-pCPT-AM, not significant (ns), P=0.9184; control+vehicle versus RSV 24 h+vehicle, ***P<0.001; RSV 24 h+vehicle versus RSV 24 h+5 µM 8-pCPT-AM, ***P<0.001 (P=0.0007). (B) Permeability of the epithelial barrier was quantified by measuring the transepithelial flux of 4 kDa FITC-conjugated dextran and normalized to control+vehicle. Data shown as mean±s.e.m., n=8 replicates, one-way ANOVA followed by Tukey's multiple comparisons test. Control+vehicle versus control+5 µM 8-pCPT-AM, not significant (ns), P=0.8495; control+vehicle versus RSV 24 h+vehicle, **P<0.01 (P=0.0036); RSV 24 h+vehicle versus RSV 24 h+5 µM 8-pCPT-AM, *P=0.0321. (C) The structure of the AJC was determined by immunostaining with antibodies towards tight junction protein ZO-1 (red) and adherens junction protein E-cadherin (green). Arrows indicate disrupted AJCs in RSV-infected cells. Images are representative of three experiments. Scale bar: 25 µm.

Fig 3: Inhibiting EPAC1/2 in human acute myeloid leukemia (AML) cell lines and in vivo‐derived Eμ‐Myc lymphoma cell lines AWestern analysis of the activated, GTP‐bound RAP1 of the indicated human AML cell lines following treatments with the EPAC1 inhibitor CE3F4 and EPAC2 inhibitor ESI‐05 for 72 h (representative of n = 2).BCellTiterGLO®‐based viability assay of MV4‐11, SHI‐1, SKM‐1, and THP‐1 human AML cells treated as indicated for 72 h. Graphs represent mean ± SEM of n = 3. Data were analyzed by one‐way ANOVA.C, DWestern analysis of PKA activity using the phosphorylated PKA substrate antibody on whole cell extracts isolated from the indicated early passage cell lines treated for 24 h with (C) H89 (n = 3) or (D) 6‐BNZ-cAMP (representatives of n = 3). Actin was used as a loading control.EWestern analysis of the activated, GTP‐bound RAP1 of the early passage CX‐5461 + EV‐resistant (CMB) cell extracts isolated following treatments with indicated EPAC inhibitors for 24 h (representative of n = 2).FWestern analysis of activated, GTP‐bound RAP1 using early passage drug‐naive (CTRL) Eμ‐Myc lymphoma cell extracts isolated following treatments with the selective EPAC activator 8‐pCPT-2‐O-Me-cAMP for 24 h (n = 1).GWestern analysis of the activated, GTP‐bound RAP1 of the early passage CX‐5461-resistant (CX) cell extracts isolated following treatments with indicated EPAC inhibitors for 24 h (n = 1).HPI exclusion analysis of the CX cells treated with CX‐5461 as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI‐05 for 48 h. Data were analyzed by paired one‐way ANOVA. Graphs represent mean ± SEM of n = 3. Black triangle: CX-5461‐resistant (CX) cells clone #14, black square: CX cells clone #20, black circle: CX cells clone #39.Data information: ns, not significant, P ≥ 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.Source data are available online for this figure.

Fig 4: Metformin inhibits cAMP‐EPAC1/2 signaling and resensitizes combination therapy‐resistant early passage Eμ‐Myc lymphoma cells to CX‐5461‐everolimus cotreatment in vivo AWestern analysis demonstrating the effects of metformin treatment for 48 h on the levels of active GTP‐bound RAP1 in CX and CMB cells (n = 3) and its quantitation.B, CProportion of green fluorescent protein (GFP)‐positive CMB (clone #8) cells in (B) lymph node and (C) spleen of transplanted C57BL/6 mice treated as indicated for 6 h on day 12 post‐transplant. Graphs represent mean ± SEM of six mice per group.DKaplan–Meier curve of C57BL/6 mice transplanted with CX‐5461‐everolimus‐resistant (CMB #8) early passage Eμ‐Myc lymphoma cells treated with vehicles (everolimus vehicle: 1% methylcellulose; CX‐5461/metformin vehicle: 25 mM NaH2PO4; n = 6); CX‐5461 (35 mg/kg every twice weekly) and everolimus (5 mg/kg daily; n = 8), metformin (400 mg/kg twice daily; n = 6), or CX‐5461, everolimus and metformin (35 mg/kg twice weekly, 5 mg/kg daily and 400 mg/kg twice daily, respectively; n = 8). Light gray: 5‐day metformin pre‐treatment period, dark gray: treatment period. Data were analyzed by a log‐rank (Mantel–Cox) test. Vehicle vs. CX-5461‐everolimus: P = 0.0006, Vehicle vs. CX-5461‐everolimus‐metformin: P = 0.0001. CX-5461‐everolimus vs. CX-5461‐everolimus‐metformin: P = 0.0003.Data information: (A) Graphs represent mean ± SEM of n = 3. Data were analyzed by Student's t‐test (A) or one‐way ANOVA (B, C). ns, not significant, P ≥ 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.Source data are available online for this figure.

Fig 5: Rap1 activity decreases during RSV infection and in the absence of cortactin. (A) 16HBE cells were infected with control medium (Con.) or RSV for the indicated period and subsequently subjected to Rap1 activity assays. Rap1-GTP was pulled down from cell lysates with GST-RalGDS RBD beads and analyzed by immunoblotting with anti-Rap1 antibodies. Rap1 was analyzed in the whole-cell lysate. GAPDH was used as the control for protein loading. (B) Quantification of immunoblots from experiments as in A. The ratio of Rap1-GTP to Rap1 was determined by densitometric analysis and plotted as normalized values versus control. Data shown as mean±s.e.m., n=3 independent experiments, repeated measures one-way ANOVA followed by Dunnett's multiple comparisons. Control versus RSV 3 h, not significant (ns), P=0.9855; control versus RSV 6 h, ns, P=0.9919; control versus RSV 18 h, *P<0.05 (P=0.0475); control versus RSV 24 h, *P<0.05 (P=0.0417). (C) WT, cortactin KO and RSV-infected cortactin KO 16HBE cells were subjected to Rap1 activity assays. Rap1-GTP was pulled down from cell lysates with GST-RalGDS RBD beads and analyzed by immunoblotting with anti-Rap1 antibodies. Rap1 was analyzed in the whole-cell lysate. GAPDH was used as the control for protein loading. (D) Quantification of immunoblots from experiments as in C. The Rap1-GTP to Rap1 ratio was determined by densitometric analysis and normalized to WT. Data shown as mean±s.e.m., n=3 replicates from three independent KO clones, one-way ANOVA followed by Tukey's multiple comparisons. WT versus cortactin KO, *P<0.05 (P=0.0411); WT versus cortactin KO+RSV, *P<0.05 (P=0.0284); cortactin KO versus cortactin KO+RSV not significant (ns), P=0.9490.