Journal: bioRxiv

Article Title: A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis and chemotherapy response

doi: 10.1101/517169

Figure Lengend Snippet: (A) Viability curves of A2780 cell lines transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2). Each experimental group was exposed to six different CDDP concentrations for 48h. Data were normalized to each untreated control, set to 100%. The data represent the mean ± SD of at least three independent experiments performed in quadruplicate at each drug concentration for each cell line analyzed. (B) Viability of A2780 cell lines transfected with pCMV6 (R-Ø) and with the overexpression vectors (R-ITF2). (C) Relative expression levels of ITF2 measured by quantitative RT-PCR, represented in Log10 scale; In each experimental group, the sensitive cell line transfected with pCMV6 plasmid was used as a calibrator. Each bar represents the combined relative expression of two independent experiments measured in triplicate. (D) β-catenin transcriptional activity was measured in A2780 cells after ITF2 overexpression and treatment with LiCl (10mM) for 24 hours, transfecting with Super8xTopFlash (Top) or Super8xFopFlash (Fop). The results show the fold induction of the Top/Fop ratio with respect to untreated cells (=1). Values represent the mean of three independent experiments measured by triplicate ± SD. (E) Expression analysis of the downstream gene DKK1 regulated by ITF2 in A2780 cell line transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2) for 24 and 72 hours. Representative images of DKK1 and GAPDH measured by RT-PCR. (F), Expression levels of DKK1 measured by qRT-PCR. Each assay was performed at least three times to confirm the results. *** p < 0.001; * p < 0.05 (Students T-test).

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, Maryland, USA) using previously described methodology .

Techniques: Transfection, Over Expression, Plasmid Preparation, Concentration Assay, Expressing, Quantitative RT-PCR, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis and chemotherapy response

doi: 10.1101/517169

Figure Lengend Snippet: (A) Correlation between ITF2 expression and HOXD9 in tumor and non-tumor samples from the complete cohort of 25 NSCLC patients. Pearson coefficient was used for linear correlation of the quantitative variables. (B-C) Effect of ITF2 overexpression on HOXD9 levels (B) Validation of the transfection efficiency of ITF2 at mRNA levels. Relative expression levels of ITF2 measured by qRT-PCR, in the cell line H23R, at 24 and 72 hours after transfectionrepresented in Log10 the 2-ΔΔCt. (C) Relative expression levels of HOXD9 measured by quantitative RT-PCR after ITF2 overexpression. For both (B) and (C) the resistant cell line transfected with pCMV6 plasmid was used as a calibrator (R-Ø). H23R cells were also transfected with ITF2 cDNA (R-ITF2). Each bar represents the combined relative expression of two independent experiments measured in triplicate. *** p < 0.001; ** p < 0.01 (Students T-test). (D) Correlation between ITF2 and HOXD9 expression levels with the overall survival analyzed in NSCLC patients selected form the RNA-seq analysis. Data represents the quantitative expression levels of the two genes measured by qRT-PCR and represented as 2-ΔΔCt for ITF2 (referred to the NLM) and 2-ΔCt for HOXD9 . (E) Survival analysis in NSCLC samples according to the mean of HOXD9 . LogRank, Breslow and Tarone-Ware test were used for comparisons and p < 0.05 was considered as a significant change in OS. (F-H) Survival analysis in 1,926 patients from the Kaplan Meier online tool (F) and the TCGA data sets of lung Adenocarcinoma (G, High=23; No Change=203) and SCC (H, High=28; No Change=441) for HOXD9 .

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, Maryland, USA) using previously described methodology .

Techniques: Expressing, Over Expression, Transfection, Quantitative RT-PCR, Plasmid Preparation, RNA Sequencing Assay

Journal: Nucleic Acids Research

Article Title: Transcription factor 4 maintains endothelial cell identity by inhibiting endothelial to mesenchymal transition

doi: 10.1093/nar/gkaf931

Figure Lengend Snippet: scRNA-seq analysis of in vitro EndoMT revealed the critical role of TCF4 as a transcriptional repressor to safeguard EC identity. ( A ) Weighted nearest-neighbor UMAP (wnnUMAP) of ECs in control (without TGFβ treatment) and TGFβ 7d (TGFβ treatment for 7 days). Each dot represents a cell. Color labels show the condition of TGFβ treatment. ( B–D ) Boxplots show the gene signature score of different gene sets in ECs of control and TGFβ 7d condition. The y -axis represents the gene set enrichment score computed using UCell software . (B) Results for the gene set “Endothelial Cell Development”. (C) Results for the gene set “Positive Regulation of Mesenchymal Cell Proliferation”. (D) Results for the gene set “Mesenchymal Cell Migration”. Those gene sets were downloaded from the MSigDB database. ( E ) A violin plot shows the TCF4 expression level in single cells of control and TGFβ 7d conditions. The y -axis represents the normalized gene expression level. ( F and G ) Violin plots show the gene signature score of the TCF OE down-regulated gene set (F) and the TCF4 KD up-regulated gene set (G) in ECs of control and TGFβ 7d conditions. The y -axis represents the gene set enrichment score computed using UCell software. ( H–K ) The wnnUMAP plots show the gene expression level of TCF4, CDH5, TGFβ2, and ACTA2 genes. Each dot represents a cell. Dots are colored by normalized gene expression levels. ( L ) The wnnUMAP plot shows the EC subgroups divided into EC-like, EndoMT, and mesenchymal-like cells. Color represents EC subgroups. ( M ) Dot plot shows the averaged expression level of TCF4, CDH5, TGFβ2, and ACTA2 genes. Dot color represents the average expression level of the gene in an EC subgroup. Dot size denotes the percentage of cells expressing the gene in the cell population. ( N ) Box plots show the gene signature score of gene sets of TCF4 KD up-regulated genes and TCF4 OE down-regulated genes. The x -axis represents the z-score scaled gene set enrichment score computed using UCell software. The y -axis represents EC subgroups. Box color represents gene sets. P -values were computed using two-sided t- test for (B), (C), (D), (F), (G), and (N). P -values were computed using two-sided Wilcoxon test for (E). *** P -values < 0.0001.

Article Snippet: Samples were incubated with a primary antibody against TCF4 (Cell Signaling Technology, #2569) or SNAI1 (Cell Signaling Technology, #3879) overnight at 4°C.

Techniques: In Vitro, Control, Software, Migration, Expressing, Gene Expression

Journal: Nucleic Acids Research

Article Title: Transcription factor 4 maintains endothelial cell identity by inhibiting endothelial to mesenchymal transition

doi: 10.1093/nar/gkaf931

Figure Lengend Snippet: Epigenetic landscape analysis proposes TCF4 as an EC identity gene. ( A ) Peak width of H3K4me3 and H3K27ac within ± 10 kb of the gene body of an individual gene locus in HUVECs. Ranks of each gene in terms of peak width for both markers are calculated, and genes are ordered by the sum of both ranks. ( B ) Signal density of individual chromatin marks at the TCF4 locus in h1ESCs, HUVECs, and normal human lung fibroblasts (NHLFs). ( C and D ) The expression level of TCF4 at individual intervals during EC differentiation detected by RNA-seq (C), and the formation of mESC–hEC heterokaryons ( D ). ( E ) Representative images of in vitro tube formation in control and TCF4 knockdown (KD) HUVECs; scale bars = 400 μm. ( F ) The number of EC tube branching points per field. ( G ) The length of the EC branching tube. ( H ) Representative wound closure images at 0, 6, and 18 h after scratching; scale bars = 400 μm. ( I ) Quantification of the distance between the two borders (dotted lines in H) in wound closure images. ( J ) NO oxide production by HUVECs. ( K ) Representative images of LDL uptake; scale bars = 200 μm. ( L ) Quantification of Ac-LDL uptake. ( M ) HUVEC proliferation. The same number of cells was used for each group. Error bars represent variation between replicates. Data are presented as mean values ± SD. n ≥ 3 biologically independent samples. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. P -values were determined by two-tailed Student's t -test.

Article Snippet: Samples were incubated with a primary antibody against TCF4 (Cell Signaling Technology, #2569) or SNAI1 (Cell Signaling Technology, #3879) overnight at 4°C.

Techniques: Expressing, RNA Sequencing, In Vitro, Control, Knockdown, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Transcription factor 4 maintains endothelial cell identity by inhibiting endothelial to mesenchymal transition

doi: 10.1093/nar/gkaf931

Figure Lengend Snippet: TCF4 depletion in ECs induces EndoMT. ( A ) qPCR results showing mRNA expression changes of EC markers (PECAM1, CDH5, and CD34) and mesenchymal markers (α-SMA, Col1a1, N-cadherin, and vimentin) in control and TCF4 KD HUVECs. ( B and C ) Representative IF staining images of CDH5 and α-SMA in control and TCF4 KD HUVECs; scale bars = 50 μm. ( D ) Western blot showing protein expression level change after TCF4 KD. ( E ) Heatmap showing DEGs between TCF4 KD and control HUVEC RNA-seq samples. Genes that pass the thresholds (FDR <0.05 and |log2foldchange| >1) are defined as either up-regulated (224) or down-regulated (416). ( F ) Heatmap showing DEGs between TCF4 overexpression (OE) and control HUVEC RNA-seq samples. ( G ) Pathways enriched in TCF4-regulated genes defined by TCF4 KD RNA-seq-based differential gene expression analysis shown in (E). ( H ) Pathways enriched in TCF4-regulated genes defined by TCF4 OE RNA-seq. ( I ) Cumulative fraction of genes plotted against TCF4 direct regulatory potential scores computed using Cistrome-GO based on TCF4 CUT&RUN peaks. Three gene sets were analyzed, naming TCF4 KD up-regulated genes (red curve), TCF4 KD down-regulated genes (blue curve), and random genes (gray curve). ( J ) Cumulative fraction of genes plotted against TCF4 direct regulatory potential scores for TCF4 OE down-regulated genes (red curve), TCF4 OE up-regulated genes (blue curve), and random genes (gray curve). Two-sided Wilcoxon rank sum test was used to compute the P -values. Data are presented as mean values ± SD. n ≥ 3 biologically independent samples. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. P -values were determined by two-tailed Student's t -test.

Article Snippet: Samples were incubated with a primary antibody against TCF4 (Cell Signaling Technology, #2569) or SNAI1 (Cell Signaling Technology, #3879) overnight at 4°C.

Techniques: Expressing, Control, Staining, Western Blot, RNA Sequencing, Over Expression, Gene Expression, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Transcription factor 4 maintains endothelial cell identity by inhibiting endothelial to mesenchymal transition

doi: 10.1093/nar/gkaf931

Figure Lengend Snippet: TCF4 modulates the TGFβ signaling pathway in EndoMT. ( A ) qPCR results showing gene expression changes of TGFβ1 after TCF4 KD in HUVECs. ( B ) ELISA results showing protein expression changes of TGFβ after TCF4 KD in HUVECs. ( C ) qPCR results showing gene expression changes of SNAI1 after TCF4 KD in HUVECs. ( D and E ) GSEA of the gene signature on differential gene expression data from TCF4 KD versus control RNA-seq. Results for gene sets of TGFβ signaling pathways from two different sources are shown. ( F and G ) Genomic tracks showing TCF4 peaks at TGFβ1 gene loci (F) and at SNAI1 gene loci (G) (highlighted in orange rectangles), along with other epigenetic signals, including DNase-seq, H3K4me1, H3K4me1, and H3K27ac. ( H–K ) qPCR results showing mRNA expression changes of TCF4 and mesenchymal markers (α-SMA, SNAI1, and Col1a1) in control, TCF4 KD, and TCF4 KD + SB431542 HUVECs. ( L ) Western blot showing changes in protein expression levels after TCF4 KD and SB431542 treatment. ( M–P ) qPCR results showing mRNA expression changes of TCF4 and mesenchymal markers (α-SMA, SNAI1, and Col1a1) in control, TCF4 KD, and TCF4 KD + SNAI1 KD HUVECs. ( Q ) Western blot showing protein expression level change after TCF4 KD and SNAI1 KD. ( R ) Genomic tracks of CUT&RUN showing the binding of TCF4 and SNAI1 at the CDH5 gene region (highlighted in orange rectangles), along with other tracks of DNase-seq and related histone modification. ( S ) SNAI1 ChIP-qPCR in both control and TCF4 KD HUVECs for the SNAI1–CDH5 binding site. ( T ) SNAI1 ChIP-qPCR in both SNAI1 OE and TCF4 OE HUVECs for the SNAI1–CDH5 binding site * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. P -values were determined by two-tailed Student's t -test.

Article Snippet: Samples were incubated with a primary antibody against TCF4 (Cell Signaling Technology, #2569) or SNAI1 (Cell Signaling Technology, #3879) overnight at 4°C.

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay, Expressing, Control, RNA Sequencing, Protein-Protein interactions, Western Blot, Binding Assay, Modification, ChIP-qPCR, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Transcription factor 4 maintains endothelial cell identity by inhibiting endothelial to mesenchymal transition

doi: 10.1093/nar/gkaf931

Figure Lengend Snippet: TCF4 overexpression alleviates TGFβ-induced EndoMT. ( A–C ) qPCR results (A) showing gene expression level changes, western blot (B) and IF staining (C) showing protein expression level change, after TGFβ1 treatment with or without TCF4 OE; scale bars = 50 μm. ( D ) Representative images of in vitro tube formation; scale bars = 400 μm. ( E ) The number of EC tube branching points per field. ( F ) The length of EC branching tubes. ( G ) qPCR results of eNOS. ( H ) NO production. ( I ) Images of Ac-LDL uptake; scale bars = 200 μm. Error bars represent variation between replicates. Data are presented as mean values ± SD. n ≥ 3 biologically independent samples. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. P -values were determined by two-tailed Student's t-t est.

Article Snippet: Samples were incubated with a primary antibody against TCF4 (Cell Signaling Technology, #2569) or SNAI1 (Cell Signaling Technology, #3879) overnight at 4°C.

Techniques: Over Expression, Gene Expression, Western Blot, Staining, Expressing, In Vitro, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Transcription factor 4 maintains endothelial cell identity by inhibiting endothelial to mesenchymal transition

doi: 10.1093/nar/gkaf931

Figure Lengend Snippet: Overexpression of TCF4 mitigates EndoMT and EC dysfunction in ECs isolated from patients with hypertension-associated heart failure. ( A–C ) qPCR results (A) showing gene expression level changes, western blot (B) and IF staining (C) showing protein expression level change, after AngII treatment with or without TCF4 OE. Scale bars = 50 μm. ( D ) Representative images of TCF4 staining. The black arrow indicates a vascular EC. Scale bars = 50 μm. ( E ) Quantification of TCF4 expression in ECs. ( F–I ) Changes in gene expression (F), cell proliferation rate (G), eNOS mRNA expression level (H), and NO production (I) after TCF4 OE in isolated ECs from heart failure patients. ( J–L ) Representative images of in vitro tube formation; scale bars = 400 μm (J), the number of EC tube branching points per field (K), and the length of EC branching tubes (L) in isolated ECs from heart failure patients with or without TCF4 OE. Error bars represent variation between replicates. Data are presented as mean values ± SD. n ≥ 3 biologically independent samples. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. P -values were determined by two-tailed Student's t -test.

Article Snippet: Samples were incubated with a primary antibody against TCF4 (Cell Signaling Technology, #2569) or SNAI1 (Cell Signaling Technology, #3879) overnight at 4°C.

Techniques: Over Expression, Isolation, Gene Expression, Western Blot, Staining, Expressing, In Vitro, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen

doi: 10.1038/s41467-022-30134-9

Figure Lengend Snippet: a DAXX degradation after infection. 293T-ACE2 cells were infected with SARS-CoV-2 at MOI 0.1. After 24 h, cells were harvested and levels of DAXX, Lamin B, HSP90, Actin, GAPDH, Tubulin, TRIM22, RIG-I and of the viral protein spike were analyzed by Western Blot. A Western Blot representative of 3 independent experiments is shown. b GRL0617 and MG132 treatments restore DAXX expression. 293T-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.1. When indicated, cells were pretreated 2 h before infection with GRL0617 (at the indicated concentrations), or with MG132 (10 µM), a proteasome inhibitor, or Masitinib (10 µM) a 3CL inhibitor. After 24 h, cells were harvested and levels of DAXX, GAPDH and of the viral protein spike were analyzed by Western Blot. A Western Blot representative of 3 independent experiments is shown. c GRL0617 treatment restores DAXX localization. 293T-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.1. 24 h post-infection, cells were labelled with Hoescht and with antibodies against dsRNA (detecting viral RNA, in green) and HA (detecting DAXX, in red). When indicated, cells were treated with 50 µM of GRL0617 at the time of infection. Scale bars correspond to 10 µm. Images are representative of 3–6 different fields from 2 independent experiments. d – f : Nsp3 induces DAXX degradation. d 293T-ACE2 cells were transfected with 1 μg of the indicated viral proteins. After 24 h, the levels of DAXX and GAPDH were analyzed by Western Blot. e 293T-ACE2 cells were transfected with the indicated amounts of Nsp3. After 24 h, the levels of DAXX and GAPDH were analyzed by Western Blot. f 293T-ACE2 cells were transfected with 1 μg of Nsp3 or of pcDNA. 6 h post transfection, cells were also, when indicated, treated with 50 µM of GRL0617. Of, 24 h after transfection, the levels of DAXX and GAPDH were analyzed by Western Blot. Western Blots representative from 2 independent experiments are shown. The quantification of band intensity for Fig. 6d–f is shown in Fig. . Source data are provided as a Source Data file.

Article Snippet: The plasmids encoding mCherry-tagged viral proteins were a gift from Bruno Antonny and ordered through Addgene: Nsp3 -mCherry (#165131); Nsp4-mCherry (#165132); Nsp6-mCherry (#165133); Nsp7-mCherry (#165134); Nsp10-mCherry (#165135); Nsp13-mCherry (#165136); Nsp14-mCherry (#165137).

Techniques: Infection, Western Blot, Expressing, Transfection

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: MIN6 cells were plated at 5 × 10 5 cells per well in 6-well plates and exposed to a TCF7L2-specific short hairpin RNA (shTCF7L2) or a scrambled shRNA (shScr) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A ) Western blots showing the TCF7L2 protein content after E 2 or P 4 treatment. ( B ) Viable cells. ( C,D ) Basal and stimulated insulin/proinsulin secretions (normalized to viable cell numbers). ( E,F ) Stimulatory indexes. ( G ) Proinsulin-to-insulin ratio. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 shTCF7L2 vs. shScr.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: shRNA, Cell Culture, Western Blot, Control

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: MIN6 cells were plated at 5 × 10 5 cells per well in 6-well plates and exposed to high glucose concentration (33.3 mM) and transfected with TCF7L2-IRES2-EGFP (OE-TCF7L2) or a control vector (CV) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A ) Western blots showing the TCF7L2 protein content after E 2 or P 4 treatment. ( B ) Viable cells. ( C , D ) Basal and stimulated insulin/proinsulin secretions (normalized to viable cell numbers). ( E,F ) Stimulatory indexes. ( G ) Proinsulin-to-insulin ratio. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 OE-TCF7L2 vs. CV.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: Concentration Assay, Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: HepG2 cells (2.5 × 10 5 cells per well) were seeded in 6-well plates and exposed to a TCF7L2-specific short hairpin RNA (shTCF7L2) or a scrambled shRNA (shScr) for 72 h, or transfected with TCF7L2-IRES2-EGFP (OE-TCF7L2) or a control vector (CV) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A,B ) 2-NBDG uptake. ( C,D ) Glucose production. ( E,F ) Western blot. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 shTCF7L2 vs. shScr or OE-TCF7L2 vs. CV.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: shRNA, Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot