Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb B: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (%), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean ± SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey's HSD; different letters indicate p < 0.05. (D) Transfection efficiency (% GFP+) of a control plasmid (pMAX-GFP) quantified by flow cytometry. (E) JMP profiler for least-squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% Cls. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection, Control, Plasmid Preparation, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb C: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (6), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean = SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey’s HSD; different letters indicate p < 0.05. (D) JMP profiler for least-squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% Cls. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb E: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (%), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean ± SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey’s HSD; different letters indicate p < 0.05. (D) JMP profiler for least- squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% CIs. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: E2F1, E2F7 and MYBL2 are highly expressed in GC tissues and correlated with the prognosis of GC patients. A , mRNA levels of E2F1, E2F7 and MYBL2 in GC tissues and normal stomach tissues in TCGA dataset. B , mRNA levels of E2F1, E2F7, and MYBL2 evaluated by RT-qPCR (n = 3) in 30 GC tissues and their paracancerous tissues. C , representative pictures of IHC for E2F1, E2F7, and MYBL2 in clinical GC samples. Scale bars: 100 μm. D , scatter plots of E2F1, E2F7, and MYBL2 IHC staining scores on 30 paired clinical samples (N = 30, T = 30). E , E2F1 WBs for 30 paired clinical samples (N = 30, T = 30). In this paper, all numbers beneath each band ( i.e. 0.67) represent the relative intensities of the WB bands, normalized to the intensities of their corresponding internal reference bands. F , quantification of E2F1 protein levels in ( E ). Top : unpaired comparisons; Bottom : paired comparisons. G – I , Kaplan Meier curves for OS ( left column), FPS ( middle column) and PPS ( right column) for patients with GC having high or low mRNA levels of E2F1 ( G ), MYBL2 ( H ), and E2F7 ( I ). J , Kaplan-Meier OS curves for GC patients with high or low ratios of E2F1/E2F7 mRNA levels ( top panel) or E2F7/E2F1 mRNA levels ( bottom panel) corresponding to OS in GC patients. In this paper, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns: not significant.

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Quantitative RT-PCR, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: Effects of knockdown of E2F1, E2F7, or MYBL2 on GC cell proliferation and apoptosis. A , protein levels of E2F1, E2F7, and MYBL2 in 5 GC cell lines and one normal gastric mucosal cell line were evaluated by WB (n = 3). In this article, all cellular WB experiments were conducted with three independent biological replicates and the numerical values beneath each band were from the single blot shown. B , changes in protein levels after E2F1, E2F7 or MYBL2 knockdown in AGS and HGC-27 cells were evaluated by WB (n = 3). C , changes in cell growth curves drawn by cell counting after E2F1, E2F7, or MYBL2 knockdown in AGS and HGC-27 cells (n = 4). D , effects of E2F1, E2F7, or MYBL2 knockdown on the colony formation ability of AGS cells were assessed by a colony formation assay (n = 3). E , changes in cell cycle distributions after E2F1, E2F7, or MYBL2 knockdown in AGS cells were evaluated by flow cytometry (n = 3). F , changes in cellular proliferation of AGS cells after knockdown of E2F1, E2F7, or MYBL2 were assessed by EdU immunofluorescence staining (n = 3). Scale bar: 100 μm. G , changes in Ki67 and PCNA protein levels after E2F1, E2F7 or MYBL2 knockdown in GC cells were evaluated by WB (n = 3). H , changes in levels of apoptosis of AGS and HGC-27 cells after E2F1, E2F7, or MYBL2 knockdown were assessed by a TUNEL assay (n = 3). Positive control group (C+) cells were treated with DNase. Scale bar: 100 μm.

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Knockdown, Cell Counting, Colony Assay, Flow Cytometry, Immunofluorescence, Staining, TUNEL Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: E2F1 upregulates E2F7 and MYBL2, whereas E2F7 downregulates E2F1 and MYBL2 in GC cells. A , correlation between E2F1, E2F7, and MYBL2 mRNA levels in GC tissues from the TCGA dataset (n = 341). B , correlation between E2F1, E2F7, and MYBL2 mRNA levels in clinical GC samples (n = 30). C , changes in mRNA levels of E2F1, E2F7 and MYBL2 in AGS and HGC-27 cells following siE2F1 or siE2F7 transient transfections were evaluated by RT-qPCR (n = 3). D , changes in E2F1, E2F7 and MYBL2 protein levels in AGS and HGC-27 cells after siE2F1 ( left panel) and siE2F7 ( right panel) transient transfections were evaluated by WB (n = 3). E , quantification of E2F1, E2F7, and MYBL2 protein levels in AGS and HGC-27 cells after siE2F1 ( left panel in ( D )). F , quantification of E2F7, E2F1 and MYBL2 protein levels in AGS and HGC-27 cells after siE2F7 ( right panel in ( D )). G , changes in mRNA levels of MYBL2 and E2F7 after E2F1 overexpression in AGS and HGC-27 cells were evaluated by RT-qPCR (n = 3), and changes in protein levels of E2F1, MYBL2, E2F7, Ki67 and PCNA after E2F1 overexpression were evaluated by WB (n = 3). H , changes in mRNA levels of MYBL2 and E2F1 after E2F7 overexpression in AGS and HGC-27 cells were evaluated by RT-qPCR (n = 3), and changes in protein levels of E2F7, MYBL2, E2F1, Ki67 and PCNA after E2F7 overexpression were evaluated by WB (n = 3).

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Transfection, Quantitative RT-PCR, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: Both E2F1 and E2F7 bind to the MYBL2 promoter and respectively activate and repress its transcription in GC cells. A , consensus binding sites of human E2F1 and E2F7. B , E2F1 and E2F7 binding to the MYBL2 and E2F1 promoters in HGC-27 cells was assessed by ChIP-qPCR (n = 3). The γ-tubulin promoter serves as a negative control. C , schematic diagram of the MYB-PGL3 plasmid containing the MYBL2 promoter sequence (−670 ∼ −170), with the E2F binding site (−212 ∼ −201) in the red box. D , changes of relative luciferase activities following overexpression of E2F1 or E2F7 in AGS and HGC-27 cells were evaluated by a dual-luciferase reporter assay (n = 3).

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Binding Assay, ChIP-qPCR, Negative Control, Plasmid Preparation, Sequencing, Luciferase, Over Expression, Reporter Assay

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: E2F1 promotes GC cell proliferation by activating MYBL2, whereas E2F7 inhibits GC cell proliferation by repressing MYBL2. A , E2F1, MYBL2, Ki67, and PCNA protein levels in tet vector + tet shNC, tet OE-E2F1 + tet shNC, tet OE-E2F1 + shMYBL2, and tet vector + tet shMYBL2 groups were evaluated by WB (n = 3). B , cell growth curves were plotted using a cell counting assay (n = 4). C , changes in colony forming ability were evaluated by a colony formation assay (n = 3, (∗∗∗∗) p < 0.0001 vs. tet OE-E2F1 + tet shNC group, #### p < 0.0001 vs. tet OE-E2F1 + tet shMYBL2 group). D , cell cycle distributions were assessed by flow cytometry (n = 3, (∗∗) p < 0.01/(∗∗∗) p < 0.001 vs. tet OE-E2F1 + tet shNC group). E , cell proliferation levels were assessed by EdU immunofluorescence assay (n = 3, (∗∗) p < 0.01 vs. tet OE-E2F1 + tet shNC group). Scale bar: 100 μm. F , E2F7, MYBL2, Ki67 and PCNA protein levels in shNC + tet shNC, shE2F7 + tet shNC, shE2F7 + tet shMYBL2, and shNC + tet shMYBL2 groups were evaluated by WB (n = 3). G , cell growth curves were plotted using a cell counting assay (n = 4). H , colony-forming abilities were evaluated by a colony formation assay (n = 3, (∗∗∗∗) p < 0.0001 vs. shE2F7 + tet shNC group, # p < 0.05 vs. shE2F7 + tet shMYBL2 group). I , cell cycle distributions were assessed by flow cytometry (n = 3, (∗∗) p < 0.01/(∗∗∗) p < 0.001 vs. shE2F7 + tet shNC group). J , cell proliferation levels were assessed by EdU immunofluorescence assay (n = 3, (∗∗) p < 0.01 vs. shE2F7 + tet shNC group). Scale bar: 100 μm.

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Plasmid Preparation, Cell Counting, Colony Assay, Flow Cytometry, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: Knockdown of MYBL2 attenuates the tumor-promoting effects of E2F1 overexpression or E2F7 knockdown ex vivo . A , representative images of nude mice and tumors stripped from mice after the injection of AGS cells of four groups induced by doxycycline: tet vector + tet shNC, tet OE-E2F1 + tet shNC, tet OE-E2F1 + shMYBL2 and tet vector + tet shMYBL2 (n = 5). L: left . R: right . B , tumor weights of xenograft tumors in nude mice (n = 5). C , growth curves of tumor xenografts in nude mice (n = 5). D , Kaplan-Meier tumor-free curves in different groups of nude mice were presented (n = 5). E , representative images of H & E staining and Ki67 IHC staining in tumor samples. Scale bar: 50 μm. The percentages of positive cells were shown in the right panel (n = 5, (∗∗) p < 0.01 vs. tet OE-E2F1 + tet shNC group, # p < 0.05 vs. tet OE-E2F1 + tet shMYBL2 group). F , apoptosis of tumor samples was assessed by a TUNEL assay. Scale bar: 50 μm. The percentages of positive cells were shown in the right panel (n = 5, (∗∗∗∗) p < 0.0001 vs. tet OE-E2F1 + tet shNC group, ## p < 0.01 vs. tet OE-E2F1 + tet shMYBL2 group). G , representative images of nude mice and tumors stripped from mice after the injection of AGS cells of four groups induced by doxycycline: shNC + tet shNC, shE2F7 + tet shNC and shE2F7 + tet shMYBL2 and shNC + tet shMYBL2 (n = 5). L: left . R: right . H , tumor weights of tumor xenografts in ( G ) (n = 5). I , growth curves of tumor xenografts in ( G ) (n = 5). J , Kaplan-Meier tumor-free curves in different groups of nude mice ( G ) were presented (n = 5). K , representative images of H & E staining and Ki67 IHC staining in tumor samples in ( G ). Scale bar: 50 μm. The percentages of positive cells were shown in the right panel (n = 5, (∗∗∗∗) p < 0.0001 vs. shNC + tet shNC group, #### p < 0.0001 vs. shE2F7 + tet shMYBL2 group). L , apoptosis of tumor samples in ( G ) was assessed by a TUNEL assay. Scale bar: 50 μm. The percentages of positive cells were shown in the right panel (n = 5).

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Knockdown, Over Expression, Ex Vivo, Injection, Plasmid Preparation, Staining, Immunohistochemistry, TUNEL Assay

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: E2F1 and E2F7 activate and repress MYBL2 to regulate cell growth through the PI3K/AKT pathway. A , oncogenic signaling pathways enriched by GSEA based on the differentially expressed genes between GC tissues with high MYBL2 expression levels and those with low MYBL2 expression levels. B , GSEA plot showing that the MYBL2-regulated genes correlate with the PI3K/AKT signaling pathway. C , the FGFR family genes among differentially expressed genes. D , PI3K, P-PI3K, AKT and P-AKT protein levels in tet vector + tet shNC, tet OE-E2F1 + tet shNC, tet OE-E2F1 + shMYBL2, and tet vector + tet shMYBL2 groups of AGS cells were evaluated by WB (n = 3, (∗∗∗) p < 0.001/(∗∗∗∗) p < 0.0001 vs. tet OE-E2F1 + tet shNC group). E , PI3K, P-PI3K, AKT and P-AKT protein levels in shNC + tet shNC, shE2F7 + tet shNC, shE2F7 + tet shMYBL2, and shNC + tet shMYBL2 groups of AGS cells were evaluated by WB (n = 3, (∗∗) p < 0.01/(∗∗∗∗) p < 0.0001 vs. shE2F7 + tet shNC group). F , PI3K, P-PI3K, AKT and P-AKT protein levels in shNC + Vector, shE2F1 + Vector, shE2F1 + OE-MYBL2, and shNC + OE-MYBL2 groups of HGC-27 cells were evaluated by WB (n = 3, (∗) p < 0.05 vs. shE2F1 + Vector group). G , PI3K, P-PI3K, AKT and P-AKT protein levels in Vector 1 + Vector 2, OE-E2F7 + Vector 2, OE-E2F7 + OE-MYBL2, and Vector 1 + OE-MYBL2 groups of HGC-27 cells were evaluated by WB (n = 3, (∗∗∗) p < 0.001 vs. OE-E2F7 + Vector two group). H , changes in MYBL2, PI3K, P-PI3K, AKT and P-AKT protein levels after MYBL2 overexpression treated with or without LY294002 in AGS cells were evaluated by WB (n = 3). I , changes in cell growth curves drawn by cell counting after MYBL2 overexpression treated with or without LY294002 in AGS cells (n = 4).

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Protein-Protein interactions, Expressing, Plasmid Preparation, Over Expression, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: Higher levels of nuclear E2F7 protein in GC cells correspond to lower mRNA levels of E2F1 and MYBL2 and lower protein levels of Ki67. A , nucleocytoplasmic distributions of E2F7 protein in adjacent tissues and GC tissues were evaluated by IHC staining (the N/C/GC panel reuses the bottom left quadrant from Fig. 1 C to highlight the specific distribution pattern). Scale bar: 50 μm. B , percentage of different E2F7 localization in adjacent tissues and GC tissues (λ 2 test, p < 0.01). C , nucleocytoplasmic distributions of E2F7 protein in 12 paired adjacent tissues and GC tissues were evaluated by WB. “∗” represented the specific band. D , statistics of the ratios of cytoplasmic E2F7 protein levels to nuclear E2F7 protein levels in ( C ). E , mRNA levels of E2F1 and MYBL2 in 30 GC samples with different nucleocytoplasmic distributions of E2F7 protein. F , representative images of IHC for Ki67 with different staining scores in GC tissues. Scale bars: 50 μm. G , statistics of Ki67 IHC staining scores in 30 GC samples with different nucleocytoplasmic distributions of E2F7 protein.

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Immunohistochemistry, Staining

Journal: The Journal of Biological Chemistry

Article Title: E2F1 and E2F7 regulate gastric cancer cell proliferation, respectively, through transcriptional activation and transcriptional repression of MYBL2

doi: 10.1016/j.jbc.2024.108027

Figure Lengend Snippet: Work model. E2F1 and E2F7 promote or inhibit cell proliferation in GC cells through direct transcriptional activation or repression of MYBL2 via PI3K/AKT signaling pathway, and form a negative feedback regulation in which E2F1 up-regulates E2F7 and E2F7 down-regulates E2F1. The nuclear protein levels of E2F7 determine the extent of its transcriptional repression and its impact on cell proliferation.

Article Snippet: Cell lysates were diluted and incubated with anti-E2F1 antibody (NBP2-67899, Novus, 5 μg), anti-E2F7 antibody (ab245655, Abcam, 5 μg), and normal rabbit IgG antibody (2729, CST, 2 μg) at 4 °C overnight.

Techniques: Activation Assay

Journal: Carcinogenesis

Article Title: Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer

doi: 10.1093/carcin/bgw020

Figure Lengend Snippet: IL-6 stimulates E2F1-dependent transcriptional activation of ILK gene expression. (A) Left, Effects of the ectopic expression of E2F1, NF-κB, and Stat3, relative to the pCMV control, on ILK promoter-driven luciferase activity in MCF-7 cells (upper panel; data are presented as mean ± SD, n = 3). Lower panel, Ectopic expression of protein was confirmed by western blot. Right, E2F1 and ILK mRNA levels in MCF-7 cells ectopically expressing E2F1 as determined by qPCR. Data are presented as mean ± SD (n = 3). (B) Left, Effects of siRNA-mediated knockdown of E2F1 on ILK promoter-driven luciferase activity (upper panel; data are presented as mean ± SD, n = 3) and ILK protein levels (lower) in MCF-7IL-6 cells. Right, E2F1 and ILK mRNA levels, as determined by qPCR, in MCF-7IL-6 cells with siRNA-mediated knockdown of E2F1. Data are presented as mean ± SD (n = 3). (C) Western blot analyses of the effects of ectopic expression of E2F1 on ILK expression in MCF-7 cells (upper panel), and siRNA-mediated knockdown of E2F1 on ILK expression in MDA-MB-231 cells (lower panel). (D) ChIP (left) and EMSA (right) analyses of the effect of exogenous IL-6 (50ng/ml) on E2F1 binding to the ILK promoter. Left, upper, Diagram of the ILK promoter showing the region containing E2F1 binding sites and the fragments amplified by the two primer pairs used for RT-PCR, of which pair #2 served as negative control. (E) EMSA analysis of the specificity of E2F1 binding to putative E2F1 binding sites on ILK promoter (-368 to -335) using MDA-MB-231 cell nuclear lysate. Lane 1 (probe-only control) shows non-specific binding and free probe. Lane 2 shows the shift after E2F1 binding. Lane 3 shows that the shift of protein-DNA complex includes ILK promoter probe by inclusion of a 50-fold molar excess of unlabeled probe. Lane 4 and 5 show that the shift of protein-DNA complex includes E2F1 by inclusion of 50-fold molar excess of E2F1 consensus probe or mutated E2F1 consensus probe, respectively.

Article Snippet: For cold competition analysis, nuclear extracts were pre-incubated with 50-fold unlabeled ILK promoter probe, E2F1 consensus binding probe (sc-2507, Santa Cruz Biotechnology, Santa Cruz, CA) or E2F1 mutant binding probe (sc-2508, Santa Cruz, CA) for 15min on ice before the addition of biotin-labeled probe.

Techniques: Activation Assay, Gene Expression, Expressing, Control, Luciferase, Activity Assay, Western Blot, Knockdown, Binding Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Carcinogenesis

Article Title: Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer

doi: 10.1093/carcin/bgw020

Figure Lengend Snippet: IL-6 activates E2F1 expression through Stat3/CyclinD1/CDK2 signaling. (A) Western blot (upper) and qPCR (lower) analyses of the differential expression of E2F1, cyclin D1, and/or ILK in four breast cancer cell lines (MCF-7, MDA-MB-468 [468], SUM-159, MDA-MB-231 [231]). Data are presented as mean ± SD (n = 3). (B) Dose-dependent effect of 24-hour treatment with exogenous IL-6 on ILK promoter-driven luciferase activity (upper) and protein levels of E2F1, cyclin D1, and ILK (lower) in MCF-7 cells. Data are presented as mean ± SD (n = 3). (C) The effect of CDK2 inhibitor SU9516 (20 μM, 48-h treatment) on E2F reporter luciferase activity (upper) and expression levels of ILK and cyclin D1 protein and phosphorylation status of CDK2 and Rb (lower) in MCF-7IL-6 cells. Data are presented as mean ± SD (n = 3). (D) Western blot analysis of the effect of the Stat3 inhibitor S3I-201 (48-hour treatment) on ILK, E2F1 and cyclin D1 expression and phosphorylation status of Stat3 and Rb in MCF-7IL-6 cells. (E) The effect of shRNA-mediated knockdown of ILK in MCF-7IL-6, MDA-MB-231 and SUM-159 cells on the expression/activation status of ILK and the IL-6 downstream signaling markers, Akt and Stat3.

Article Snippet: For cold competition analysis, nuclear extracts were pre-incubated with 50-fold unlabeled ILK promoter probe, E2F1 consensus binding probe (sc-2507, Santa Cruz Biotechnology, Santa Cruz, CA) or E2F1 mutant binding probe (sc-2508, Santa Cruz, CA) for 15min on ice before the addition of biotin-labeled probe.

Techniques: Expressing, Western Blot, Quantitative Proteomics, Luciferase, Activity Assay, Phospho-proteomics, shRNA, Knockdown, Activation Assay

Journal: Carcinogenesis

Article Title: Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer

doi: 10.1093/carcin/bgw020

Figure Lengend Snippet: Validation of the ILK-Akt-NF-κB-IL-6 signaling loop. (A) Left, confirmation of GFP, ILK, CA-Akt and RelA protein levels in MDA-MB-231TRE-shILK ectopically expressing CA-Akt, GFP-RelA or GFP, in the presence or absence of doxycycline (Dox) by Western blotting. Ectopic expression of CA-Akt and RelA reversed the suppressive effects of doxycycline-induced ILK knockdown on NF-κB luciferase reporter activity (middle) and IL-6 mRNA expression (right) in MDA-MB-231TRE-shILK cells. Data are presented as mean ± SD (n = 3). (B) Ectopic expression of CA-Akt and RelA restored the proliferation rate of ILK-knockdown MDA-MB-231TRE-shILK cells. Data are presented as mean ± SD (n = 3). +, doxycycline-treated. (C) Suppressive effects of doxycycline (Dox)-induced ILK knockdown on the growth of subcutaneous MDA-MB-231TRE-shILK xenograft tumors. Data are presented as mean ± SD (n = 6). (D) Ectopic expression of CA-Akt and RelA restored the growth of ILK-knockdown (+Dox) MDA-MB-231TRE-shILK tumors to a level comparable to that of ILK-intact controls (-Dox). Mean tumor volumes at 21 days after tumor cell injection are shown. Data are presented as mean ± SD (-Dox, n = 6; +Dox, n = 10; +Dox/+Akt, n = 10; +Dox/+RelA, n = 7). (E) Schematic diagram depicting the role of the Stat3-cyclin D1/CDK2-E2F1-ILK axis in mediating the IL-6-NF-κB feedback loop.

Article Snippet: For cold competition analysis, nuclear extracts were pre-incubated with 50-fold unlabeled ILK promoter probe, E2F1 consensus binding probe (sc-2507, Santa Cruz Biotechnology, Santa Cruz, CA) or E2F1 mutant binding probe (sc-2508, Santa Cruz, CA) for 15min on ice before the addition of biotin-labeled probe.

Techniques: Biomarker Discovery, Expressing, Western Blot, Knockdown, Luciferase, Activity Assay, Injection