Journal: Journal for immunotherapy of cancer

Article Title: IGF1 receptor inhibition amplifies the effects of cancer drugs by autophagy and immune-dependent mechanisms.

doi: 10.1136/jitc-2021-002722

Figure Lengend Snippet: Figure 3 PPP induces autophagy via IGF1R/AKT signaling. (A, B) U2OS-GFP-LC3 cells were incubated in the absence of FBS overnight and were further treated with PPP (10 μM) or torin1 (300 nm) in the presence or absence of IGF1 (10 nm) for 6 hours. After fixation, GFP-LC3 dots were quantified in B. Scale bar equals 10 μm. Data are means±SD of three replicates (**P<0.01, vs DMSO/Ctr, Student’s t-test). (C) Immunoblot exploration of the IGF1R signal pathway. After U2OS cells were incubated in the absence of FBS overnight, then the cells were treated with PPP (10 μM) together with or without IGF1 (10 nm) for 6 hours. Proteins were separated by SDS-PAGE and parallel immunoblots of pIGF1R (Tyr1135/1136), IGF1R, pAkt (Ser473), Akt, pmTOR (Ser2448), mTOR, pp70 (THR389), p70, pTFEB (Ser211), TFEB, LC3-II and were performed in parallel instances and β-actin (ACTB) was used as loading control (C). (D, E) U2OS wild-type (WT) or IGF1R knockout cells were treated with PPP (10 μM) for 6 H. SDS-PAGE and immunoblots of IGF1R, LC3 and ACTB were performed (D), band intensities of LC3-II and ACTB were assessed, and their ratio (LC3-II/ACTB) was calculated (online supplemental figure S4). In parallel U2OS-GFP-LC3 WT or IGF1R knockout cells were treated with PPP (10 μM) for 6 hours then the cells were fixed and GFP-LC3 dots were quantified by microscopy (F). Representative images are shown in E and scale bar equals 10 μm. Data are means±SD of three replicates (***P<0.001 vs untreated control; ##P<0.01 vs WT; Tukey’s multiple comparisons test). (G–I) U2OS cells were transfected with constitutive active AKTT308D/S473D and were treated with PPP (10 μM) for 6 hours. SDS-PAGE and immunoblots of pAKT, AKT, LC3 and ACTB were performed (G), band intensities of LC3-II and ACTB were assessed, and their ratio (LC3-II/ACTB) was calculated (online supplemental figure S4). In parallel U2OS-GFP-LC3 WT or AKTT308D/S473D-expressing cells were treated with PPP (10 μM) for 6 H then the cells were fixed and GFP-LC3 dots were quantified by microscopy (I). Representative images are shown in H and scale bar equals 10 μm. Data are means±SD of three replicates (**P<0.01 vs untreated control; ##P<0.01 vs WT; Tukey’s multiple comparisons test). (J–K) U2OS cells stably expressing GFP-AKT or GFP-AKTR25C were incubated in absence of FBS overnight, then the cells were treated with IGF1 (10 nm) or PPP (10 μM) combined with IGF1 (10 nm). After 5 min, the membrane translocation of GFP-AKT was detected by microscopy (J) and the membrane intensity of AKT was measured (K). Scale bar equals 10 μm. Data are means±SD of three replicates (***P<0.001 vs DMSO/Ctr, Student’s t-test). IGF1, FBS, fetal bovine serum; IGF1, insulin-like growth factor-1; PPP, picropodophyllin; SDS_PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Article Snippet: Cells were seeded, let adhere for 24 hours, and following transfected with the CRISPR- Cas9 plasmid U6gRNA- Cas92A- RFP targeting IGF1R (50- ATGATGCGATT CTTCGACG-30) or a plasmid coding for AKT carrying the T308D/S473D mutation (#14751, Addgene, Watertown, Massachusetts, USA), according to the manufacturer’s advice.

Techniques: Incubation, Western Blot, SDS Page, Control, Knock-Out, Microscopy, Transfection, Expressing, Stable Transfection, Membrane, Translocation Assay, Polyacrylamide Gel Electrophoresis

Journal: Journal for immunotherapy of cancer

Article Title: IGF1 receptor inhibition amplifies the effects of cancer drugs by autophagy and immune-dependent mechanisms.

doi: 10.1136/jitc-2021-002722

Figure Lengend Snippet: Figure 4 IGF1R signaling correlates with immunosuppressive markers and decreased survival in breast cancer. (A–I) The expression of CD163, FOXP3 and CD8 as well as the phosphorylation of IGF1R and the dot formation of LC3 were quantified in paraffin-embedded biopsies obtained from 49 triple-negative breast cancer patients by ImageJ after staining with specific antibodies. Representative images of phosphorylated-IGF1R, dotted LC3B, and CD163, FOXP3 and CD8 expression are shown in A. The scale bar indicates 100 μm. Correlation analyses (determined by the Spearman method) of the analyzed parameters for each patient are depicted in B–E. (F–I) Kaplan-Meier survival analysis of patients with biomarker-positive and biomarker- negative immunohistochemistry staining. IGF1R, insulin-like growth factor-1 receptor.

Article Snippet: Cells were seeded, let adhere for 24 hours, and following transfected with the CRISPR- Cas9 plasmid U6gRNA- Cas92A- RFP targeting IGF1R (50- ATGATGCGATT CTTCGACG-30) or a plasmid coding for AKT carrying the T308D/S473D mutation (#14751, Addgene, Watertown, Massachusetts, USA), according to the manufacturer’s advice.

Techniques: Expressing, Phospho-proteomics, Staining, Biomarker Discovery, Immunohistochemistry

Journal: Journal for immunotherapy of cancer

Article Title: IGF1 receptor inhibition amplifies the effects of cancer drugs by autophagy and immune-dependent mechanisms.

doi: 10.1136/jitc-2021-002722

Figure Lengend Snippet: Figure 6 IGF1R inhibition improves the anticancer efficacy of immunotherapy. (A) Schematic overview of the in vivo treatment of murine lung cancer Tc1 cells with oxaliplatin (OXA), anti-PD-1 antibody and PPP, alone or in combination. (B–E) Growth kinetic of Tc1 cells evolving in immunocompetent C57BL/6 mice, treated as indicated in (A). When tumors became palpable, mice received systemic intraperitoneal injection of PPP alone or together with OXA or PD-1 blocker. Data ae depicted as (B) growth curves (mean±SD); (C) tumor size distributions at day 24; (D) individual tumor growth curves of mice treated with OXA and PPP, combined or not with PD-1 blockade. The generation of immunological memory was assessed in cured animals by rechallenge with MCA205 and Tc1. Individual tumor growth curves are depicted (E). Data were analyzed with TumGrowth. n≥ 6 for mice in each group. (*P<0.05 or ns, not statistically significant vs OXA; #P<0.05 or ns, not statistically significant vs OXA+PD-1 blocker, Student’s t-test, survival plots in online supplemental figure S7). (F) Schematic overview of the in vivo treatment of murine fibrosarcoma MCA205 cells with OXA, anti-PD-1 antibody and the selective of IGF1R inhibitor linsitinib (Lins), alone or in combination. IGF1R, insulin-like growth factor-1 receptor; PPP, picropodophyllin.

Article Snippet: Cells were seeded, let adhere for 24 hours, and following transfected with the CRISPR- Cas9 plasmid U6gRNA- Cas92A- RFP targeting IGF1R (50- ATGATGCGATT CTTCGACG-30) or a plasmid coding for AKT carrying the T308D/S473D mutation (#14751, Addgene, Watertown, Massachusetts, USA), according to the manufacturer’s advice.

Techniques: Inhibition, In Vivo, Injection

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex-mediated stimulation on INSR/IGF1R and ERK1/2. HEK293 cells were pretreated with dimethyl sulfoxide (linsitinib [−]) or with linsitinib (linsitinib [+]) and subsequently stimulated with Pas2r12, Pas2r12–EGFP, Pas2r12–IgG, or insulin for 2 min ( A – C ) or 10 min ( D – F ). Panels A and D show representative Western blot images. Panels B and E display the phosphorylation levels of INSR/IGF1R (pINSR/pIGF1R); panels C and F show the phosphorylation levels of ERK1/2 (pERK1/2); all values were normalized to GAPDH. Phosphorylation levels were analyzed and compared with the solvent control (DMEM only, linsitinib [−]) using Student’s t -test. For each treatment condition, linsitinib (+) was compared with the corresponding linsitinib (−) condition. Statistically significant differences compared with the control are indicated on the bar graphs; significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Statistical comparisons were performed against control cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05 and ** p < 0.01. N = 4.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Solvent, Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R and INSR knockdown on the Pas2r12-mediated cytosolic delivery of EGFP. Western blot analyses ( A – C ) and confocal laser scanning microscopy images ( D , E ). Graphs B and C show IGF1R and INSR expression levels, respectively, normalized to GAPDH. Cells analyzed with confocal laser scanning microscopy in panel A were analyzed by Western blot ( A – C ). ( D ) Pas2r12-mediated cytosolic delivery of EGFP in knockdown cells, with EGFP fluorescence shown in green, and ( E ) percentage of cells exhibiting cytosolic EGFP delivery. Scale bars represent 20 μm. Statistical comparisons were performed against siNC cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 4.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Confocal Laser Scanning Microscopy, Expressing, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Assessment of IGF1R overexpression. ( A ) Verification of IGF1R expression levels in HEKI cells. ( B ) Relative levels of IGF1R expression normalized to GAPDH based on the data in panel A. Values for IGF1R/GAPDH are expressed compared with parental HEK293 cells (set to 1). Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3. ( C ) Subcellular localization of IGF1R is shown in green; nuclei were counterstained with Hoechst 33342 (blue). Scale bars represent 20 μm.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Over Expression, Expressing

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in IGF1R-overexpressing cells. ( A ) HEKI#66 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Level of phosphorylated INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R, normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated HEKI#66 control (linsitinib [−]), and, for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Solvent, Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R overexpression on the Pas2r12-mediated cytosolic delivery of EGFP. ( A ) Representative confocal images showing the cellular uptake of Pas2r12–EGFP in IGF1R-overexpressing (HEKI) cells. Scale bars represent 20 μm. ( B ) Relative levels of the cytosolic delivery efficiency of EGFP in HEKI cells compared with parental HEK293 cells (set to 100%). Merged images show EGFP fluorescence (green) and differential interference contrast. Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). ** p < 0.01. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Over Expression, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in INSR-overexpressing cells. ( A ) IN#1 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Phosphorylation of INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R were normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated IN#1 control (linsitinib [−]), and for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot, Solvent, Control

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a SKBR3, pool2, BT474, and HR20 cells cultured at normal condition were collected and subjected to western blot analyses of p-IGF-1R, IGF-1R, IRS1, p-Akt (T308) , p-Akt (S473) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin. b The levels of IGF1 and IGF2 in conditioned medium (CM) were measured by ELISA, **** p < 0.0001. c Pool2 or HR20 cells stably transfected with control shRNA (sh-Con) or IRS1 -targeting shRNAs (sh-1#, sh-2#) were treated with Herceptin at indicated concentrations for 72 h. Cell viability was evaluated by MTS assays, Pool2 sh-1#: ** p = 0.0021, **** p < 0.0001, sh-2#: ** p = 0.0096, **** p < 0.0001, HR20 sh-1#: * p = 0.0154, ** p = 0.0015, **** p < 0.0001, sh-2#: ** p = 0.0072, ** p = 0.0034, **** p < 0.0001. d Pool2 or HR20 cells with IRS1 deletion via CRISPR-Cas9 gene editing (sg-Con vs sg-1# and sg-2#) were treated with Herceptin at indicated concentrations for 72 h. Cell viability was evaluated by MTS assays, Pool2 sg-1#: *** p = 0.0002, **** p < 0.0001, sg-2#: * p = 0.0235, ** p = 0.0017, **** p < 0.0001, HR20 sg-1#: *** p = 0.0003, **** p < 0.0001, sg-2#: ** p = 0.0023, **** p < 0.0001. e Downregulation of IRS1 was achieved with either specific shRNAs (sh-Con vs sh-1# and sh-2#) or CRISPR-Cas9 gene editing (sg-Con vs sg-1# and sg-2#) in Pool2 or HR20 cells. The expression of IRS1, p-IGF-1R, IGF-1R, p-Akt (T308) , p-Akt (S473) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin was measured by western blot assays. f SKBR3 and BT474 cells were stably transfected with control vector (pLEX-Con or Con) or IRS1-overexpressing vector (pLEX-IRS1 or IRS1) followed by treatment with rhIGF2 (80 ng/ml) in combination with Herceptin at indicated concentrations for 72 h. Cell viability was evaluated by MTS assays, SKBR3 IRS1 + IGF2: ** p = 0.0059, *** p = 0.0002, *** p = 0.0003, BT474 IRS1 + IGF2: ** p = 0.0038, *** p = 0.0004. n = 3 biological independent samples ( b – d , f ). Data are presented as mean values ± SEM ( b – d , f ). Statistical significance was determined by a two-tailed Student’s t -test ( b – d , f ). Data show a representative of three independent experiments ( a , e ). All data are provided in the Source Data file.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, shRNA, CRISPR, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a SKBR3 cells were treated with rhIGF2 at indicated concentrations for 6 h. The expression of p-IGF-1R, IGF-IR, p-Akt (S473) , p-Akt (T308) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin was examined by western blot assays. b SKBR3 cells were transfected with a control empty vector (Con) or the same vector containing an IRS1 cDNA (IRS1), followed by rhIGF2 treatment for 6 h. The expression of indicated proteins was examined by western blot assays. c SKBR3 cells were transfected with control shRNA (sh-Con) or specific FOXO3a shRNAs (sh-1# and sh-2#) followed by rhIGF2 treatment for 6 h. The expression of indicated proteins was examined by western blot assays. d SKBR3 cells with IRS1 gene deletion via CRISPR-Cas9 (sg-Con vs sg-1# and sg-2#) were treated by rhIGF2 treatment for 6 h. The expression of indicated proteins were examined by western blot assays. Data show a representative of three independent experiments ( a – d ). All data are provided in the Source Data file.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, shRNA, CRISPR

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a, b SKBR3 or BT474 cells were treated with vehicle (Mock) or Rapamycin (10 μM, Rapa) in combination with rhIGF2 at indicated concentrations for 24 h. The expression of p-Akt (S473) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, IRS1, PPP3CB, and β-actin was examined by western blot assays ( a ); The expression levels of miR-128-3p and miR-30a-5p were detected by qRT-PCR ( b ), **** p < 0.0001. c SKBR3 or BT474 cells were treated with a series of phosphatase inhibitors as Cantharidic acid (0.5 μM), Endothall (1 μM), RK-682 (10 μM), Cypermethrin (1 μM), Deltamethrin (1 μM), RWJ-60475 (2 μM), Tyrphostin 8 (10 μM), CinnGel (1 μM), BML-260 (10 μM), or BN-82002 (5 μM) in combination with rhIGF2 at indicated concentrations for 24 h. The expression of p-FOXO3a, FOXO3a, and β-actin was analyzed by western blot assays. d SKBR3 cells were treated with rhIGF2 at indicated concentrations for 24 h. The expression of PPP3CB, PPP3CA, PPP3CC, PPP3R1, PPP3R2, and β-actin was examined by western blot assays. e , f SKBR3 or BT474 cells with specific knockdown of PPP3CB by shRNAs (sh-Con vs sh-1# and sh-2#) were treated with rhIGF2 (80 ng/ml) for 24 h. The expression of PPP3CB, p-FOXO3a, FOXO3a and β-actin were examined by western blot assays ( e ); The enrichment of FOXO3a at the promoters of miR-128-3p and miR-30a-5p were detected by ChIP-qPCR ( f ), **** p < 0.0001. n = 3 biological independent samples ( b , f ). Data are presented as mean values ± SEM ( b , f ). Statistical significance was determined by a two-tailed Student’s t -test ( b , f ). Data show a representative of three independent experiments ( a , c , d , e ). All data are provided in the Source Data.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a The expression levels of miR-128-3p and miR-30a-5p in SKBR3, pool2, BT474, or HR20 cells were measured by qRT-PCR, **** p < 0.0001. b Pool2 or HR20 cells were transfected with miR-128-3p or/and miR-30a-5p mimics. The expression of IRS1, p-FOXO3a, FOXO3a, and β-actin was examined by western blot assays. c Pool2 cells were transfected with miRNA mimics in combination with pLEX-IRS1 followed by Herceptin treatment at indicated concentrations for 72 h (left). SKBR3 cells were transfected miRNA inhibitors in combination with IRS1 shRNA, and then treated with 80 ng/ml rhIGF2 along with Herceptin (right). Cell viability was evaluated by MTS assays. Pool2 Mimics: ** p = 0.0022, *** p = 0.0003, **** p < 0.0001, Mimics+pLEX-IRS1: *** p = 0.0001, **** p < 0.0001, SKBR3 Inhibitors+IGF2: ** p = 0.0072, ** p = 0.0035, **** p < 0.0001, Inhibitors+IGF2 + sh-IRS1: ** p = 0.0079, *** p = 0.0002, **** p < 0.0001. d The expression levels of miR-193a-5p in SKBR3, pool2, BT474, and HR20 cells were detected by qRT-PCR, **** p < 0.0001. e Pool2 or HR20 cells were transfected with a miR-193a-5p mimic. SKBR3 or BT474 were transfected with a miR-193a-5p inhibitor. IGF2 levels in the CM were measured by ELISA. f Pool2 or HR20 cells were treated with vehicle (Mock) or WAY-600 (1 μM, WAY). The expression of p-Akt (S473) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin was examined by western blot assays (left); the expression levels of miR-128-3p, miR-30a-5p and miR-193a-5p were detected by qRT-PCR (right), **** p < 0.0001. g Pool2 or HR20 cells were treated with WAY-600 for 24 h. The enrichment of FOXO3a at miR-193a-5p promoter was examined by ChIP-qPCR assays, **** p < 0.0001. n = 3 biological independent samples ( a , c – e , g ). Data are presented as mean values ± SEM ( a , c – e , g ). Statistical significance was determined by a two-tailed Student’s t -test ( a , c – e , g ). Data show a representative of three independent experiments ( b , f ). All data are provided in the Source Data.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, shRNA, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a SKBR3, pool2, BT474, and HR20 cells cultured at normal condition were collected and subjected to western blot analyses of p-IGF-1R, IGF-1R, IRS1, p-Akt (T308) , p-Akt (S473) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin. b The levels of IGF1 and IGF2 in conditioned medium (CM) were measured by ELISA, **** p < 0.0001. c Pool2 or HR20 cells stably transfected with control shRNA (sh-Con) or IRS1 -targeting shRNAs (sh-1#, sh-2#) were treated with Herceptin at indicated concentrations for 72 h. Cell viability was evaluated by MTS assays, Pool2 sh-1#: ** p = 0.0021, **** p < 0.0001, sh-2#: ** p = 0.0096, **** p < 0.0001, HR20 sh-1#: * p = 0.0154, ** p = 0.0015, **** p < 0.0001, sh-2#: ** p = 0.0072, ** p = 0.0034, **** p < 0.0001. d Pool2 or HR20 cells with IRS1 deletion via CRISPR-Cas9 gene editing (sg-Con vs sg-1# and sg-2#) were treated with Herceptin at indicated concentrations for 72 h. Cell viability was evaluated by MTS assays, Pool2 sg-1#: *** p = 0.0002, **** p < 0.0001, sg-2#: * p = 0.0235, ** p = 0.0017, **** p < 0.0001, HR20 sg-1#: *** p = 0.0003, **** p < 0.0001, sg-2#: ** p = 0.0023, **** p < 0.0001. e Downregulation of IRS1 was achieved with either specific shRNAs (sh-Con vs sh-1# and sh-2#) or CRISPR-Cas9 gene editing (sg-Con vs sg-1# and sg-2#) in Pool2 or HR20 cells. The expression of IRS1, p-IGF-1R, IGF-1R, p-Akt (T308) , p-Akt (S473) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin was measured by western blot assays. f SKBR3 and BT474 cells were stably transfected with control vector (pLEX-Con or Con) or IRS1-overexpressing vector (pLEX-IRS1 or IRS1) followed by treatment with rhIGF2 (80 ng/ml) in combination with Herceptin at indicated concentrations for 72 h. Cell viability was evaluated by MTS assays, SKBR3 IRS1 + IGF2: ** p = 0.0059, *** p = 0.0002, *** p = 0.0003, BT474 IRS1 + IGF2: ** p = 0.0038, *** p = 0.0004. n = 3 biological independent samples ( b – d , f ). Data are presented as mean values ± SEM ( b – d , f ). Statistical significance was determined by a two-tailed Student’s t -test ( b – d , f ). Data show a representative of three independent experiments ( a , e ). All data are provided in the Source Data file.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, shRNA, CRISPR, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a SKBR3 cells were treated with rhIGF2 at indicated concentrations for 6 h. The expression of p-IGF-1R, IGF-IR, p-Akt (S473) , p-Akt (T308) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin was examined by western blot assays. b SKBR3 cells were transfected with a control empty vector (Con) or the same vector containing an IRS1 cDNA (IRS1), followed by rhIGF2 treatment for 6 h. The expression of indicated proteins was examined by western blot assays. c SKBR3 cells were transfected with control shRNA (sh-Con) or specific FOXO3a shRNAs (sh-1# and sh-2#) followed by rhIGF2 treatment for 6 h. The expression of indicated proteins was examined by western blot assays. d SKBR3 cells with IRS1 gene deletion via CRISPR-Cas9 (sg-Con vs sg-1# and sg-2#) were treated by rhIGF2 treatment for 6 h. The expression of indicated proteins were examined by western blot assays. Data show a representative of three independent experiments ( a – d ). All data are provided in the Source Data file.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, shRNA, CRISPR

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a SKBR3 or BT474 cells were transfected with miR-128-3p or/and miR-30a-5p inhibitor (upper) or transfected with miR-128-3p or/and miR-30a-5p mimics (bottom). The expression of IRS1 and β-actin was examined by western blot assays. b SKBR3 or BT474 cells were transfected with miR-128-3p or/and miR-30a-5p inhibitor followed by rhIGF-2 treatment at indicated concentration for 24 h. The expression of IRS1 and β-actin was examined by western blot assays. c SKBR3 or BT474 cells were transfected with FOXO3a shRNA followed by rhIGF-2 treatment at indicated concentration for 24 h. The expression levels of miR-128-3p and miR-30a-5p were measured by qRT-PCR, **** p < 0.0001. d A schematic representation of FOXO3a binding sites within the 2 kb putative promoters of miR-128-3p and miR-30a-5p. The first base of the precursors of miR-128-3p and miR-30a-5p is defined as ‘+1’. e SKBR3 or BT474 cells were treated with rhIGF-2 at indicated concentrations for 24 h. The enrichment of FOXO3a at miR-128 or miR-30a promoter was evaluated by ChIP-qPCR. The chromatin was precipitated with an anti-FOXO3a antibody. The precipitated chromatin was then analyzed by qRT-PCR with primers specific for the putative FOXO3a binding sites, **** p < 0.0001. n = 3 biological independent samples ( c , e ). Data are presented as mean values ± SEM ( c , e ). Statistical significance was determined by a two-tailed Student’s t -test ( c , e ). Data show a representative of three independent experiments ( a , b ). All data are provided in the Source Data file.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Expressing, Western Blot, Concentration Assay, shRNA, Quantitative RT-PCR, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a, b SKBR3 or BT474 cells were treated with vehicle (Mock) or Rapamycin (10 μM, Rapa) in combination with rhIGF2 at indicated concentrations for 24 h. The expression of p-Akt (S473) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, IRS1, PPP3CB, and β-actin was examined by western blot assays ( a ); The expression levels of miR-128-3p and miR-30a-5p were detected by qRT-PCR ( b ), **** p < 0.0001. c SKBR3 or BT474 cells were treated with a series of phosphatase inhibitors as Cantharidic acid (0.5 μM), Endothall (1 μM), RK-682 (10 μM), Cypermethrin (1 μM), Deltamethrin (1 μM), RWJ-60475 (2 μM), Tyrphostin 8 (10 μM), CinnGel (1 μM), BML-260 (10 μM), or BN-82002 (5 μM) in combination with rhIGF2 at indicated concentrations for 24 h. The expression of p-FOXO3a, FOXO3a, and β-actin was analyzed by western blot assays. d SKBR3 cells were treated with rhIGF2 at indicated concentrations for 24 h. The expression of PPP3CB, PPP3CA, PPP3CC, PPP3R1, PPP3R2, and β-actin was examined by western blot assays. e , f SKBR3 or BT474 cells with specific knockdown of PPP3CB by shRNAs (sh-Con vs sh-1# and sh-2#) were treated with rhIGF2 (80 ng/ml) for 24 h. The expression of PPP3CB, p-FOXO3a, FOXO3a and β-actin were examined by western blot assays ( e ); The enrichment of FOXO3a at the promoters of miR-128-3p and miR-30a-5p were detected by ChIP-qPCR ( f ), **** p < 0.0001. n = 3 biological independent samples ( b , f ). Data are presented as mean values ± SEM ( b , f ). Statistical significance was determined by a two-tailed Student’s t -test ( b , f ). Data show a representative of three independent experiments ( a , c , d , e ). All data are provided in the Source Data.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a The expression levels of miR-128-3p and miR-30a-5p in SKBR3, pool2, BT474, or HR20 cells were measured by qRT-PCR, **** p < 0.0001. b Pool2 or HR20 cells were transfected with miR-128-3p or/and miR-30a-5p mimics. The expression of IRS1, p-FOXO3a, FOXO3a, and β-actin was examined by western blot assays. c Pool2 cells were transfected with miRNA mimics in combination with pLEX-IRS1 followed by Herceptin treatment at indicated concentrations for 72 h (left). SKBR3 cells were transfected miRNA inhibitors in combination with IRS1 shRNA, and then treated with 80 ng/ml rhIGF2 along with Herceptin (right). Cell viability was evaluated by MTS assays. Pool2 Mimics: ** p = 0.0022, *** p = 0.0003, **** p < 0.0001, Mimics+pLEX-IRS1: *** p = 0.0001, **** p < 0.0001, SKBR3 Inhibitors+IGF2: ** p = 0.0072, ** p = 0.0035, **** p < 0.0001, Inhibitors+IGF2 + sh-IRS1: ** p = 0.0079, *** p = 0.0002, **** p < 0.0001. d The expression levels of miR-193a-5p in SKBR3, pool2, BT474, and HR20 cells were detected by qRT-PCR, **** p < 0.0001. e Pool2 or HR20 cells were transfected with a miR-193a-5p mimic. SKBR3 or BT474 were transfected with a miR-193a-5p inhibitor. IGF2 levels in the CM were measured by ELISA. f Pool2 or HR20 cells were treated with vehicle (Mock) or WAY-600 (1 μM, WAY). The expression of p-Akt (S473) , Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, and β-actin was examined by western blot assays (left); the expression levels of miR-128-3p, miR-30a-5p and miR-193a-5p were detected by qRT-PCR (right), **** p < 0.0001. g Pool2 or HR20 cells were treated with WAY-600 for 24 h. The enrichment of FOXO3a at miR-193a-5p promoter was examined by ChIP-qPCR assays, **** p < 0.0001. n = 3 biological independent samples ( a , c – e , g ). Data are presented as mean values ± SEM ( a , c – e , g ). Statistical significance was determined by a two-tailed Student’s t -test ( a , c – e , g ). Data show a representative of three independent experiments ( b , f ). All data are provided in the Source Data.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, shRNA, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a The expression of PPP3CB, p-STAT6, STAT6, p-Src, Src, and β-actin in the indicated cells was analyzed by western blot assays (left). The expression levels of PPP3CB mRNA were measured by qRT-PCR (right), **** p < 0.0001. b Pool2 or HR20 cells with PPP3CB overexpression were treated with Herceptin for 72 h (top). SKBR3 or BT474 cells with specific knockdown of PPP3CB were treated with Herceptin for 72 h (bottom). Cell viability was evaluated by MTS assays, SKBR3 sh-1#: ** p = 0.008, ** p = 0.0013, *** p = 0.0002, sh-2#: ** p = 0.0019, **** p < 0.0001, BT474 sh-1#: ** p = 0.0038, *** p = 0.0002, *** p = 0.0001, sh-2#: *** p = 0.0003, *** p = 0.0001, **** p < 0.0001. c Pool2 or HR20 cells were transfected with control vector (Control) or PPP3CB-overexpressing vector (PPP3CB). The expression of PPP3CB, p-FOXO3a, FOXO3a, IRS1, and β-actin was examined by western blot assays (left). IGF2 levels in the CM were detected by ELISA (right). d The expression levels of miR-128-3p, miR-30a-5p, and miR-193a-5p in PPP3CB-overexpressing pool2 or HR20 cells were measured by qRT-PCR, **** p < 0.0001. e Pool2 or HR20 cells were treated with vehicle (Mock) or entinostat (1 μM, Ent.) for 48 h. The expression of PPP3CB, p-FOXO3a, FOXO3a, IRS1, and β-actin was examined by western blots. f , g Pool2 or HR20 cells were transfected with control shRNA (Con or sh-Con) or specific STAT6 shRNAs (sh-2# or sh-4#). The expression of STAT6, PPP3CB, p-FOXO3a, FOXO3a, IRS1, and β-actin was examined by western blots ( f ); the enrichment of HDAC1 at PPP3CB promoter was determined by ChIP-qPCR ( g ), **** p < 0.0001. h Total protein extracts of inducated cells were subjected to IP using an anti-HDAC1 antibody or control IgG, followed by western blot analysis of HDAC1 or p-STAT6. i Pool2 or HR20 cells treated with vehicle (Mock) or SU6656 (10 μM) were examined by western blot analysis. n = 3 biological independent samples ( a , b , c , d , g ). Data are presented as mean values ± SEM ( a , b , c , d , g ). Statistical significance was determined by a two-tailed Student’s t -test ( a , b , c , d , g ). All data are provided in the Source Data.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a Pool2/sh-Con or pool2/sh-IRS1 cells were subcutaneously inoculated into the armpit of female Balb/C athymic nude mice to generate tumor xenografts. When the tumor size reached ~100 mm 3 , the mice were randomly grouped and received intraperitoneal (i.p.) injection of PBS or Herceptin (10 mg/kg) ( n = 5) once every 5 days. The tumor growth was measured every 5 days (left). At the experimental endpoint, the tumor weights were measured (right), **** p < 0.0001. b Pool2 cells with control sgRNA vector (sg-Con) or IRS1 gene specific sgRNA (sg-IRS1) were subcutaneously inoculated into the armpit of female Balb/C athymic nude mice to generate xenograft tumors. When the tumor sizes reached ~100 mm 3 , the mice were randomly grouped and received i.p. injection of Herceptin (10 mg/kg) or PBS ( n = 5) once every 5 days. The tumor growth was measured every 5 days (left). At the experimental endpoint, the tumor weights were measured (right), **** p < 0.0001. c , d The serum and tumor tissues were obtained from 23 HER2-positive breast cancer patients with a good response to Herceptin-containing treatments and 17 HER2-positive breast cancer patients with a poor response to Herceptin-containing treatments. The levels of IGF2 in the serum were detected by ELISA ( c ), **** p < 0.0001; The expression of IRS1, PPP3CB, and p-FOXO3a in the tumors was examined by Immunohistochemistry (IHC) assays ( d ). Data are presented as mean values ± SEM ( c ). Statistical significance was determined by a two-tailed Student’s t -test ( a – c ). e Kaplan–Meier analyses of overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival (DMFS) in breast cancer patients with low versus high expression of PPP3CB . The survival curves were shown with the hazard ratio with 95% confidence intervals and logrank P -values.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Injection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Treatment with 17β-Estradiol Reduced Body Weight and the Risk of Cardiovascular Disease in a High-Fat Diet-Induced Animal Model of Obesity

doi: 10.3390/ijms18030629

Figure Lengend Snippet: The cardiac survival and apoptosis biomarker analysis of 60 days of different doses of 17β-estradiol treatments (0, 4, 8, 12 μg/kg/day) for high-fat diet-fed male C57B mice ( n = 6 in each group). ( A ) The hearts protein samples Western blotting analysis results of ERβ, p-IGF1R, IGF1R, p-PI3K, PI3K, p-Akt, Akt, caspase 3, and β-actin. ( B ) The normalized protein expression of ERβ/β-actin. ( C ) The normalized protein expression of p-IGF1R/β-actin. ( D ) The normalized protein expression of p-PI3K/β-actin. ( E ) The normalized protein expression of p-Akt/β-actin. ( F ) The normalized protein expression of caspase 3/β-actin. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared with indicated group.)

Article Snippet: Membranes were blocked with 3% BSA in Tris-buffered saline (TBS) solution with 0.05% Tween-20 and incubated with primary antibodies, including antibodies against β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), caspase-3 (sc-7148, Santa Cruz Biotechnology, Dallas, TX, USA), ERα (sc-7202, Santa Cruz Biotechnology, Dallas, TX, USA), ERβ (sc-8974, Santa Cruz Biotechnology, Dallas, TX, USA), p-GATA4 (sc-32823-R, Santa Cruz Biotechnology, Dallas, TX, USA), p-Akt (sc-7985, Santa Cruz Biotechnology, Dallas, TX, USA), Akt (sc-5298, Santa Cruz Biotechnology, Dallas, TX, USA), p-PI3K (sc-12929, Santa Cruz Biotechnology, Dallas, TX, USA), PI3K (sc-423, Santa Cruz Biotechnology, Dallas, TX, USA), p-IGF1R (sc-101704, Santa Cruz Biotechnology, Dallas, TX, USA), IGF1R (sc-463, Santa Cruz Biotechnology, Dallas, TX, USA), p-JAK (sc-21870, Santa Cruz Biotechnology, Dallas, TX, USA), p-Erk5 (sc-1284, Santa Cruz Biotechnology, Dallas, TX, USA), IL-6 (sc-7290, Santa Cruz Biotechnology, Dallas, TX, USA), and STAT3 (sc-483, Santa Cruz Biotechnology, Dallas, TX, USA), at 4 °C overnight.

Techniques: Biomarker Discovery, Western Blot, Expressing

Journal: Experimental Biology and Medicine

Article Title: Nuclear factor E2-related factor 2 knockdown enhances glucose uptake and alters glucose metabolism in AML12 hepatocytes

doi: 10.1177/1535370217694435

Figure Lengend Snippet: Antibodies used for Western blot analysis

Article Snippet: The ratios of the mean values of protein level in AML12 cells between two selected groups are listed in Supplementary Table 2. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Company Reference Dilution Nrf2 Santa Cruz Sc-722 1:1000 HO-1 Abcam ab52947 1:1000 NQO1 Proteintech 11451-1-AP 1:1000 p-EIF2α S51 Millipore 04-342 1:1000 EIF2α Proteintech 11233-1-AP 1:1000 IL-1β Ruiying Biological RLT2322 1:1000 TNF-α Ruiying Biological RLM3477 1:1000 p-NF-κB p65 S276 Ruiying Biological RLP0187 1:1000 MMP2 Ruiying Biological RLT2798 1:1000 MMP9 Ruiying Biological RLT1892 1:1000 FGF21 Abcam ab171941 1:1000 AMPKα Ruiying Biological RLT0215 1:1000 Sirt1 Cell signaling Q96E86 1:1000 PGC-1α Abcam ab54481 1:1000 UCP1 Abcam ab23841 1:1000 Glut-4 Ruiying Biological RLT1930 1:1000 IGF-1R Ruiying Biological RLT2282 1:1000 FOXO1 Ruiying Biological RLT1757 1:1000 p-AKT S473 Ruiying Biological RLP0006 1:1000 AKT Ruiying Biological RLT0178 1:1000 GSK3α/β Proteintech 22104-1-AP 1:500 p-GSK3α/β Y279/Y216 Signalway 11002 1:500 Gapdh Santa Cruz Sc420485 1:1000 Open in a separate window Antibodies used for Western blot analysis Statistical analysis The data are presented as the mean ± SEM for the number of replicates indicated.

Techniques: Western Blot