Fig 1: AKT is involved in MUC4 depletion-related GEM resistance. (A) A heatmap showed the relative changes in phosphorylation of different proteins from resistant cells (SNU-1196-GR and SSP-25-GR) versus parental cells (SNU-1196 and SSP-25) and from SP-25-GR cells transfected with shRNAs specific to MUC4 (shMUC4 #1) versus those transfected with shRNAs specific to LacZ (shLacZ). Red pixels: upregulated expression; blue pixels: downregulated expression. (B) Western blots showing the protein levels of phosphorylated AKT, total AKT, phosphorylated ERK, and total ERK in human CCA cell lines. a-Tubulin was used as the loading control. (C) Western blots showing the protein levels of phosphorylated AKT, total AKT, phosphorylated ERK, and total ERK in murine CCA cell lines. Actin was used as the loading control. (D) Western blots showing the protein levels of MUC4, phosphorylated AKT, total AKT, phosphorylated ERK, and total ERK in SNU-1196-GR and SSP-25-GR cells transfected with shRNAs against MUC4 (shMUC4 #1 and #2) or LacZ (shLacZ). a-Tubulin was used as the loading control. (E) Western blots showing the protein levels of AKT1 and AKT2 in SNU-1196-GR cells transfected with shRNAs against AKT1 (shAKT1 #1 and #2), AKT2 (shAKT2 #1 and #2) or LacZ (shLacZ). a-Tubulin was used as the loading control. (F) Upper: The cell viability in SNU-1196-GR cells transfected with shRNAs against AKT1 (shAKT1 #1 and #2), AKT2 (shAKT2 #1 and #2), or LacZ (shLacZ) in various concentrations of GEM. Lower: The IC50 values (means ± SDs) were from three independent experiments. **, P < 0.005 by Student's t-test. (G) Western blots showing the levels of PARP1 and MUC4 in SSP-25-GR cells transfected with shRNAs specific to AKT1 (shAKT1 #1 and #2), AKT2 (shAKT2 #1 and #2) or LacZ (shLacZ). The cells were treated with 500 nM GEM (+) or DMSO (-) for 48 hours. a-Tubulin was used as the loading control. (H) Western blots showing the protein levels of MUC4, phosphorylated AKT, total AKT, phosphorylated ERK, and total ERK in MUC4-overexpressing SNU-1196 cells. (I) Western blots showing the protein levels of AKT1 and AKT2 in MUC4-overexpressing SNU-1196 (SNU-1196/ MUC4) cells transfected with shRNAs against AKT1 (shAKT1 #1 and #2), AKT2 (shAKT2 #1 and #2) or LacZ (shLacZ). Actin was used as the loading control. (J) Right: The cell viability in MUC4-overexpressing SNU-1196 cells transfected with shRNAs against AKT1 (shAKT1 #1 and #2), AKT2 (shAKT2 #1 and #2) or LacZ (shLacZ) in various concentrations of GEM. Left: The IC50 values (means ± SDs) were from three independent experiments. **, P < 0.005 by Student's t-test.
Fig 2: AKT inhibitors in combination with GEM or afatinib decreased cell survival in GR sublines and MUC4-overexpressing CCA. (A) Left: cell viability in various concentrations of GEM. SNU-1196-GR cells were cultured in the absence (DMSO) or presence of 2 µM MK-2206 or 2 µM capivasertib and treated with various concentrations of GEM for 72 hours. Right: The IC50 values (means ± SDs) were from three independent experiments. **, P < 0.005 by Student's t-test. (B) Left: The viability of SNU-1196-GR cells treated with various concentrations of MK-2206 and GEM for 72 hours. Right: The CI values for the combination of GEM and MK-2206 in SNU-1196-GR cells. The ED50 and ED75 values (means ± SDs) were from three independent experiments. ED, effective dose. (C), (D) The cell viability in SNU-1196 cells expressing the vector alone (C, SNU-1196/vector) or MUC4 (D, SNU-1196/MUC4) in various concentrations of MK-2206 and GEM for 72 hours. (E) The CI values for the combination of GEM and MK-2206 in SNU-1196 cells expressing the vector alone (black) or MUC4 (green). The ED50 and ED75 values (means ± SDs) were from three independent experiments. ED, effective dose. **, P < 0.005 by Student's t-test. (F) Western blots showing the protein levels of PARP1, phosphorylated AKT, total AKT, phosphorylated BAX, and total BAX in SNU-1196-GR cells treated with 2 µM MK-2206 (+), 1 µM GEM (+) or DMSO (-) for 48 hours. a-Tubulin was used as the loading control. (G) Left: Representative images of the colony formation assay. SUN-1196 cells (500 cells/well) were seeded in a six-well plate for 17 days and then cultured in the absence or presence of 5 µM MK-2206 (+), 50 nM GEM (+), 100 nM GEM (++), or DMSO (-) for another four days. Right: The values (means ± SDs) were from three independent experiments; * P<0.05, ** P<0.01 by Student's t-test. NS: not significant. (H) Western blots showing the protein levels of phosphorylated HER3, total HER3, AKT1 and AKT2 in SNU-1196-GR cells transfected with shRNAs against AKT1 (shAKT1 #1 and #2), AKT2 (shAKT2 #1 and #2) or LacZ (shLacZ). Actin was used as the loading control. (I) Western blots showing the protein levels of phosphorylated HER3, total HER3, and MUC4 in SNU-1196-GR cells transfected with shRNAs against MUC4 (shMUC4 #1 and #2) or LacZ (shLacZ). a-Tubulin was used as the loading control. (J) Left: The viability of SNU-1196-GR cells treated with various concentrations of MK-2206 and afatinib for 72 hours. Right: The CI values for the combination of MK-2206 and afatinib in SNU-1196-GR cells. The ED50 and ED75 values (means ± SDs) were from three independent experiments. ED, effective dose. (K) Western blots showing the protein levels of phosphorylated HER3, total HER3, phosphorylated AKT, total AKT, phosphorylated BAX, and total BAX in SNU-1196-GR cells treated with 2 µM MK-2206 (+), 2 µM capivasertib (+), 1 µM GEM (+) or DMSO (-) for 48 hours. a-Tubulin was used as the loading control. (L) Left: Representative images of the colony formation assay. SUN-1196-GR cells (500 cells/well) were seeded in a six-well plate for 17 days and then cultured in the absence or presence of 10 µM MK-2206 (+), 10 µM capivasertib (+), 2 µM afatinib (+), 5 µM afatinib (++), or DMSO (-) for another four days. Right: The values (means ± SDs) were from three independent experiments; * P<0.05, ** P<0.01 by Student's t-test. NS: not significant
Fig 3: MUC4 conferred GEM resistance in CCA cells. (A) A heatmap showed the relative mRNA expression of MUC genes from resistant sublines (SNU-1196-GR and SSP-25-GR) compared with that of their parental cells (SNU-1196 and SSP-25). Red pixels: upregulated expression; blue pixels: downregulated expression. (B) The relative mRNA levels of MUC1, MUC4, and MUC16 (n=3). The values (means ± SDs) are presented as the fold-change relative to the level in parental cells (SNU-1196 and SSP-25). *, P < 0.05; **, P < 0.005 by Student's t test. (C) Upper: western blots showing the protein level of MUC4 in whole-cell lysates. a-Tubulin was used as the loading control. Lower: The expression of MUC4 in conditioned media (CM) was detected by an ELISA kit in human CCA cells (n=3). *, P < 0.05; **, P < 0.005 by Student's t test. (D) Western blots showing the protein level of MUC4 in three pairs of murine CCA sublines. a-Tubulin was used as the loading control. (E) MUC4 was depleted by shRNAs (shMUC4 #1 and #2) in SNU-1196-GR cells. Western blots showing the knockdown efficacy of SNU-1196-GR cells transfected with shRNAs specific to MUC4 (shMUC4 #1 and #2) or LacZ (shLacZ). (F) Left: cell viability in various concentrations of GEM. Right: The IC50 values (means ± SDs) were from three independent experiments. **, P < 0.005 by Student's t-test. (G) Western blots showing the levels of PARP1 and MUC4 in SSP-25-GR cells transfected with shRNAs specific to MUC4 (shMUC4 #1 and #2) or LacZ (shLacZ). The cells were treated with 500 nM GEM (+) or DMSO (-) for 48 hours. a-Tubulin was used as the loading control. (H) MUC4 was depleted by shRNA transfection (shMUC4 #1 and #2) in M4-GR cells. Western blots showing the knockdown efficacy in M4-GR cells transfected with shRNAs specific to MUC4 (shMUC4 #1 and #2) or LacZ (shLacZ). (I) Left: cell viability in various concentrations of GEM. Right: the IC50 values (means ± SDs) from three independent experiments. **, P < 0.005 by Student's t-test. (J) Western blots showing the levels of MUC4 in MUC4-overexpressing SNU-1196 and SSP-25 cells. Actin was used as the loading control. (K) Upper: cell viability in various concentrations of GEM. Lower: the IC50 values (means ± SDs) were from three independent experiments. The p values from Student's t-test are shown in the table.
Fig 4: The knockdown of HER2 reduced GEM sensitivity in GR- and MUC4-overexpressing cells. (A) Western blots showing the protein levels of phosphorylated HER2 and total HER2 in SNU-1196, SNU-1196-GR, SSP-25, and SSP-25-GR cells. α-Tubulin was used as the loading control. (B) Western blots showing the protein levels of phosphorylated EGFR, biotinylated EGFR, total EGFR, phosphorylated HER2, biotinylated EGFR, total HER2, and MUC4 in SSP-25-GR cells transfected with shRNAs against MUC4 (shMUC4 #1 and #2) or LacZ (shLacZ). α-Tubulin was used as the loading control. (C) Western blots showing the protein level of HER2 in SNU-1196-GR cells transfected with shRNAs against HER2 (shHER2 #1 and #2) or LacZ (shLacZ). α-Tubulin was used as the loading control. (D) Right: The cell viability in SNU-1196-GR cells transfected with shRNAs against HER2 (shHER2 #1 and #2) or LacZ (shLacZ) in various concentrations of GEM. Left: The IC50 values (means ± SDs) were from three independent experiments. *, P < 0.05; **, P < 0.005 by Student's t test. (E) Western blots showing the protein level of HER2 in MUC4-expressing SNU-1196 cells transfected with shRNAs against HER2 (shHER2 #1 and #2) or LacZ (shLacZ). Actin was used as the loading control. (F) Left: The cell viability in MUC4-expressing SNU-1196 cells transfected with shRNAs against HER2 (shHER2 #1 and #2) or LacZ (shLacZ) in various concentrations of GEM. Right: The IC50 values (means ± SDs) were from three independent experiments. **, P < 0.005 by Student's t test.
Fig 5: MUC4-AKT1 axis-mediated upregulation of hENT1 decreased GEM sensitivity in CCA. (A) Western blots showing the protein level of hENT1 in SSP-25, SSP-25-GR, SNU-1196, and SNU-1196-GR. α-Tubulin was used as the loading control. (B) Western blots showing the protein level of hENT1 in M3, M3-GR, M4, and M4-GR. α-Tubulin was used as the loading control. (C) Western blots showing the protein levels of hENT1 and MUC4 in SNU-1196 cells expressing the vector alone or in MUC4 cells. (D) Western blots showing the protein levels of hENT1, MUC4, and AKT1 in SSP-25-GR cells transfected with shRNAs against MUC4 (shMUC4 #1 and #2), AKT1 (shAKT1 #1 and #2) or LacZ (shLacZ). α-Tubulin was used as the loading control. (E) Western blots showing the protein level of hENT1 in SSP-25-GR cells receiving 2 μM MK-2206, 2 μM capivasertib, or DMSO treatment for 24 hours. α-Tubulin was used as the loading control. (F) Western blots showing the protein level of hENT1 in CCC-GR cells receiving 5 μM MK-2206, 10 μM capivasertib, or DMSO treatment for 24 hours. α-Tubulin was used as the loading control. (G) Western blots showing the protein level of hENT1 in SSP-25 cells transfected with shRNAs against hENT1 (shhENT1 #1 and #2) or LacZ (shLacZ). α-Tubulin was used as the loading control. (H) Left: The cell viability in SSP-25 cells transfected with shRNAs against hENT1 (shhENT1 #1 and #2) or LacZ (shLacZ) in various concentrations of GEM. Right: The IC50 values (means ± SDs) were from three independent experiments. *, P < 0.05; **, P < 0.005 by Student's t-test.
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