human her2 breast cancer cell lines skbr3  (ATCC)

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: Human HER2-positive breast cancer cell lines SKBR3 and BT474 were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Control, 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: Human HER2-positive breast cancer cell lines SKBR3 and BT474 were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, Transfection, Control, 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: Human HER2-positive breast cancer cell lines SKBR3 and BT474 were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Transfection, Expressing, Western Blot, Concentration Assay, shRNA, Quantitative RT-PCR, Binding Assay, ChIP-qPCR, 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: Human HER2-positive breast cancer cell lines SKBR3 and BT474 were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Knockdown, ChIP-qPCR, 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: Human HER2-positive breast cancer cell lines SKBR3 and BT474 were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, shRNA, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, 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: Human HER2-positive breast cancer cell lines SKBR3 and BT474 were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

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