human her2 breast cancer cell lines skbr3  (ATCC)

Journal: Thoracic Cancer

Article Title: Galectin‐3 enhances trastuzumab resistance by regulating cancer malignancy and stemness in HER2 ‐positive breast cancer cells

doi: 10.1111/1759-7714.14474

Figure Lengend Snippet: Galectin‐3 promoted cancer malignancy of HER2‐positive breast cancer cells. Western blot analysis of galectin‐3, HER2, and β‐actin in (a) pcDNA‐NC, pcDNA‐ LGALS3 , SKBR3, and SKBR3 cells treated with 100 ng/mL r‐Gal3 for 3 days; (b) JIMT1 cells transfected with siRNA‐NC, siRNA‐533, or siRNA‐571; and (c) JIMT1 cells treated with 0, 10, or 20 μg/mL GB1107 for 3 days. Cell viability curves for (d) pcDNA‐NC, pcDNA‐ LGALS3 , SKBR3, and SKBR3 cells treated with 100 ng/mL r‐Gal3 for 3 days; and (e) siRNA‐NC, siRNA‐533, siRNA‐571, and JIMT1 cells treated with 10 μg/mL GB1107 for 3 days. (f) Healing assays; (g) colony formation assays; and (h) transwell assays in pcDNA‐NC, pcDNA‐ LGALS3 , siRNA‐NC, and siRNA‐533 cells. The significant differences are indicated by asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The human HER2‐positive breast cancer cell lines SKBR3 (trastuzumab‐sensitive) and JIMT1 (trastuzumab‐resistant) were obtained from the American Type Culture Collection and maintained in McCoy's 5A medium and Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific), respectively.

Techniques: Western Blot, Transfection

Journal: Thoracic Cancer

Article Title: Galectin‐3 enhances trastuzumab resistance by regulating cancer malignancy and stemness in HER2 ‐positive breast cancer cells

doi: 10.1111/1759-7714.14474

Figure Lengend Snippet: Galectin‐3 activated Notch1 signaling pathway and promoted the cancer cell stemness of HER2‐positive breast cancer cells. (a) Immunofluorescence images of galectin‐3 (red) and DAPI (blue) in SKBR3‐CT cells, SKBR3‐OE cells, JIMT‐CT cells, and JIMT‐KO cells. (b) Western blot analysis of Notch1 pathway proteins (Notch1, NICD, HES1, and HEY1) and stemness biomarkers (CD24, CD44, CD133, Nanog, and E‐cadherin); β‐actin was used as a loading control. (c) Mammosphere formation in SKBR3‐OE, SKBR3‐CT, JIMT1‐KO, and JITM1‐CT cells. Representative images (top) and quantitative data (bottom) of mammosphere formation are shown. Scale bars, 100 μm. The significant differences are indicated by asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The human HER2‐positive breast cancer cell lines SKBR3 (trastuzumab‐sensitive) and JIMT1 (trastuzumab‐resistant) were obtained from the American Type Culture Collection and maintained in McCoy's 5A medium and Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific), respectively.

Techniques: Immunofluorescence, Western Blot, Control

Journal: Thoracic Cancer

Article Title: Galectin‐3 enhances trastuzumab resistance by regulating cancer malignancy and stemness in HER2 ‐positive breast cancer cells

doi: 10.1111/1759-7714.14474

Figure Lengend Snippet: Galectin‐3 affected the sensitivity of HER2‐positive breast cancer cells to trastuzumab. Cell viability curves and inhibition rate of (a) SKBR3‐CT and SKBR3‐OE cells and (b) JIMT1‐CT and JIMT1‐KO cells treated with trastuzumab for 72 hours. (c) Cell viability curves and inhibition rate of JIMT1‐CT cells treated with GB1107 for 72 hours. (d) Cell viability curves of JIMT1‐CT cells treated with GB1107 and/or trastuzumab. (e) Drug interaction analysis chart in JIMT1‐CT cells. The significant differences are indicated by asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The human HER2‐positive breast cancer cell lines SKBR3 (trastuzumab‐sensitive) and JIMT1 (trastuzumab‐resistant) were obtained from the American Type Culture Collection and maintained in McCoy's 5A medium and Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific), respectively.

Techniques: Inhibition

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