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. 2022 Sep 20;13:5506. doi: 10.1038/s41467-022-33037-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — a The protein levels and half-life times of wild-type and mutant SMAD4 in BxPC3-WT and BxPC3-Mut stably transfected cells. Data are shown as the mean ± SD from three biologically independent samples (n = 3). b Different SMAD4 and TRIM33 levels of mutant and wild-type SMAD4 with DTLL induction in BxPC3-WT and BxPC3-Mut stably transfected cells. Data are shown as the mean ± SD from three biologically independent samples (n = 3). ‘a’ indicates p < 0.05 with comparison SMAD4 of BxPC3-Mut versus BxPC3-WT cells, ‘b’ represents p < 0.05 with comparison TRIM33 of AsPC-1 versus MIA PaCa-2 cells. When treated with DTLL, p < 0.001 for SMAD4 at all time points except for 0, 0.5, and 1 h, p < 0.001 for TRIM33 at 1, 2, 6, 8, 12, and 24 h. c Distinct protein levels and half-life times of mutant and wild-type SMAD4 with DTLL induction. Data are shown as the mean ± SD from three biologically independent samples (n = 3). ‘a’ indicates p < 0.05 with comparison of BxPC3-Mut versus BxPC3-WT cells. When treated with CHX + DTLL, p < 0.001 for SMAD4 at all time points except for 0, 0.25 and 0.5 h. d Different cell signaling pathways affected by mutant and wild-type SMAD4 with DTLL induction. Data are shown as the mean ± SD from three biologically independent samples (n = 3). In BxPC3-Mut cells, p < 0.001 (‘GEM + DTLL’versus Control) for SMAD4, p-SMAD2/SMAD2, SMAD7, TGFβ and p-AKT/AKT; p < 0.001 (‘GEM + DTLL’versus GEM) for SMAD4, p-SMAD2/SMAD2, TGFβ, p-EGFR/EGFR and p-AKT/AKT; p < 0.001 (DTLL versus Control) for SMAD4, p-SMAD2/SMAD2, SMAD7 and p-AKT/AKT; p < 0.001 (DTLL versus GEM) for SMAD4, p-SMAD2/SMAD2, p-EGFR/EGFR and p-AKT/AKT. In BxPC3-WT cells, p < 0.001 (‘GEM + DTLL’versus Control) for TGFβ; p < 0.001 (DTLL versus GEM) for p-EGFR/EGFR. e Cell cycle-related proteins were determined by Western blot assays in BxPC3-Mut and BxPC3-WT stably transfected cells after treatments with 2 µM gemcitabine, 0.1 nM DTLL or its combination at 37 °C for 4 h. Data are shown as the mean ± SD from three biologically independent samples (n = 3). In BxPC3-Mut cells, p < 0.001 (‘GEM + DTLL’versus Control) for Cyclin D3 and p27; p < 0.001 (DTLL versus Control) for Cyclin D3 and p21; p < 0.001 (DTLL versus GEM) for p21 and p27. In BxPC3-WT cells, p < 0.001 (‘GEM + DTLL’versus Control) for Cyclin D3, Cyclin B1, CDK2, p-CDC2, p-Weel, p21, p27, Cyclin E2 and CDK4; p < 0.001 (‘GEM + DTLL’versus GEM) for Cyclin D3, CDK2, p-Weel, p27, Cyclin E2 and CDK4; p < 0.001 (‘GEM + DTLL’versus DTLL) for CDK2, p21, Cyclin E2 and CDK4; p < 0.001 (DTLL versus Control) for Cyclin D3, CDK2, p-Weel, p27, Cyclin E2 and CDK4; p < 0.001 (DTLL versus GEM) for Cyclin D3, CDK2, p21and p27; p < 0.001 (GEM versus Control) for CDK2, p21, p27and Cyclin E2. f TRIM33, NF-κB and apoptosis-related proteins were determined by Western blot assays in BxPC3-Mut and BxPC3-WT stably transfected cells after treatment with gemcitabine and DTLL. Data are shown as the mean ± SD from three biologically independent samples (n = 3). In BxPC3-WT cells, p < 0.001 (‘GEM + DTLL’versus Control) for SMAD4, NF-kB p50, p-NF-kB p65/NF-kB p65, Cleaved Caspase 8, FADD, MCL1, BAX, BCL2/BAX and MCL1/BAX; p < 0.001 (‘GEM + DTLL’versus GEM) for SMAD4, NF-kB p50, p-NF-kB p65/NF-kB p65, Cleaved Caspase 8, FADD; p < 0.001 (‘GEM + DTLL’versus DTLL) for SMAD4 and FADD; p < 0.001 (DTLL versus Control) for SMAD4, p-NF-kB p65/NF-kB p65, Cleaved Caspase 8, BAX, BCL2/BAX and MCL1/BAX; p < 0.001 (DTLL versus GEM) for SMAD4, p-NF-kB p65/NF-kB p65 and Cleaved Caspase 8; p < 0.001 (GEM versus Control) for p-NF-kB p65/NF-kB p65 and MCL1/BAX. In BxPC3-WT cells, p < 0.001 (‘GEM + DTLL’versus Control) for TRIM33, NF-kB p50, BCL2, MCL1, and MCL1/BAX; p < 0.001 (‘GEM + DTLL’versus GEM) for NF-kB p50, BCL2, MCL1 and MCL1/BAX; p < 0.001 (‘GEM + DTLL’versus DTLL) for NF-kB p50; p < 0.001 (DTLL versus Control) for Cleaved Caspase 8, MCL1, and MCL1/BAX; p < 0.001 (DTLL versus GEM) for Cleaved Caspase 8 and MCL1. g The mRNA levels of SMAD4, TRIM33, P50, P65, FADD, Bcl-2, MCL1, and BAX were determined in BxPC3-Mut and BxPC3-WT cells after treatments with gemcitabine, DTLL and their combination by using qRT-PCR. Data are shown as the mean ± SD from three biologically independent samples (n = 3). In BxPC3-WT cells, p < 0.001 (‘GEM + DTLL’versus Control) for NF-kB p50, NF-kB p65, TRIM 33, FADD, BcL2, MCL1, and Bax; p < 0.001 (‘GEM + DTLL’versus GEM) for NF-kB p50, NF-kB p65, TRIM 33, MCL1and Bax; p < 0.001 (‘GEM + DTLL’versus DTLL) for NF-kB p65 and MCL1; p < 0.001 (DTLL versus Control) for SMAD4, NF-kB p50, NF-kB p65, TRIM 33, BcL2, MCL1, and Bax; p < 0.001 (DTLL versus GEM) for SMAD4, NF-kB p50, TRIM 33, BcL2, MCL1, and Bax. In BxPC3-Mut cells, p < 0.001 (‘GEM + DTLL’versus Control) for SMAD4, NF-kB p50, NF-kB p65, TRIM 33, FADD and Bax; p < 0.001 (‘GEM + DTLL’versus GEM) for SMAD4, NF-kB p50, NF-kB p65, TRIM 33, FADD, BcL2, MCL1, and Bax; p < 0.001 (‘GEM + DTLL’versus DTLL) for SMAD4, NF-kB p65, FADD, and Bax; p < 0.001 (DTLL versus Control) for SMAD4, NF-kB p50, NF-kB p65, FADD, and Bax; p < 0.001 (DTLL versus GEM) for SMAD4, NF-kB p50, NF-kB p65, FADD, BcL2, MCL1, and Bax; p < 0.001 (GEM versus Control) for SMAD4, FADD, BcL2, MCL1, and Bax. h Specific interactions of SMAD4 with NF-κB and TRIM33 in BxPC3-EV, BxPC3-WT, and BxPC3-Mut stably transfected cells. The results were obtained from three biologically independent experiments. i Different protein interactions of mutant or wild-type SMAD4 with DTLL induction in BxPC3-WT and BxPC3-Mut stably transfected cells. The results were obtained from three biologically independent experiments. j Different proteomic and gemcitabine pharmacokinetic profiles in mutant and wild-type SMAD4 cells after treatment with GEM, DTLL or both. Data are shown as the mean ± SD from three biologically independent samples (n = 3). For d–g, ‘a’ indicates p < 0.05 as compared with the control, ‘b’, p < 0.05 compared with the GEM group and ‘c’, p < 0.05 compared with the DTLL group. One-way ANOVA with Bonferroni post hoc test was used in d–g; Paired-samples t-test was two-sided and used in b and c. All specific p values are presented in the Source Data file and only significant values are shown in figures. Source data are provided in the Source Data file.