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Sino Biological human her2/erbb2 transcript variant 1 gene orf cdna clone expression plasmid
Human Her2/Erbb2 Transcript Variant 1 Gene Orf Cdna Clone Expression Plasmid, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/her2+expression+vector/Human+HER2%2FERBB2+transcript+variant+1+Gene+ORF+cDNA+clone+expression+plasmid/custom%40hg10004-ut%4010%2E1101%2F2025%2E05%2E02%2E651174
Average 95 stars, based on 9 article reviews

human her2/erbb2 transcript variant 1 gene orf cdna clone expression plasmid - by Bioz Stars, 2026-09

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Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells
Article Snippet: .. The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological. .. The control siRNA (cat. no. sc-37007) and siRNA for leucine-rich repeat-containing G-protein coupled receptor 4 ( LGR4 ; cat. no. sc-62558) were purchased from Santa Cruz Biotechnology, Inc.

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Fig. 1. Specific binding of the extracellular domain of HER2 and the anti-HER2 affibody was detected as Nluc activity. (A) Schematic illustration of the IFNAR1/2 reconstitution system (IFNARRS). Proteins to be examined for their interaction are labeled as X and Y. X and Y were fused to the transmembrane and intracellular domains (TM-IN) of IFNAR1 and IFNAR2, respectively. When X and Y bind to each other, IFNAR1-TM-IN and IFNAR2-TM-IN come near and activate TYK2/JAK1 and STAT1/STAT2/IRF9 to induce Nluc activities via ISREs. NlucP is only activated by the specific binding of X and Y via elimination of IFNα-dependent activation with knock-out of the endogenous IFNAR1 gene. (B) An extracellular domain of HER2 and an anti-HER2 affibody were fused to the transmembrane and intracellular domains of IFNAR1 and IFNAR2, as model binding proteins. (C) Of the four possible combinations, two ([1] + [4] and [3] + [2]) caused significant increases in Nluc activities when transfected into HEK293T cells with the 6xISRE-NlucP plasmid. After 36 h, Nluc luciferase activities were measured (mean ± SD of three independent experiments). (D) Different numbers of ISRE were placed in the promoter of NlucP (SI Appendix, Fig. S1B) and transfected into HEK293T cells in a 96-well plate (Negative: [3] + [4] and Positive: [3] + [2]). After 36 h, the Nluc assay was performed. Asterisks indicate significant difference compared each Positive to Negative (*P < 0.01, **P < 0.0001). IFNAR1 and IFNAR2, interferon alpha, and beta receptor subunits 1 and 2; EX, extracellular domain; TM, transmembrane region; IN, intracellular domain; JAK, Janus kinase; TYK2, tyrosine kinase 2; STAT, signal transducer and activator of transcription; IRF9, interferon regulatory factor 9; ISRE, interferon-stimulated response element; NlucP, a destabilized form of Nluc with a PEST sequence; PM, plasma membrane; HER2, human EGFR- related 2; and affibody, Af.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Development of a highly sensitive platform for protein-protein interaction detection and regulation of T cell function.

doi: 10.1073/pnas.2318190121

Figure Lengend Snippet: Fig. 1. Specific binding of the extracellular domain of HER2 and the anti-HER2 affibody was detected as Nluc activity. (A) Schematic illustration of the IFNAR1/2 reconstitution system (IFNARRS). Proteins to be examined for their interaction are labeled as X and Y. X and Y were fused to the transmembrane and intracellular domains (TM-IN) of IFNAR1 and IFNAR2, respectively. When X and Y bind to each other, IFNAR1-TM-IN and IFNAR2-TM-IN come near and activate TYK2/JAK1 and STAT1/STAT2/IRF9 to induce Nluc activities via ISREs. NlucP is only activated by the specific binding of X and Y via elimination of IFNα-dependent activation with knock-out of the endogenous IFNAR1 gene. (B) An extracellular domain of HER2 and an anti-HER2 affibody were fused to the transmembrane and intracellular domains of IFNAR1 and IFNAR2, as model binding proteins. (C) Of the four possible combinations, two ([1] + [4] and [3] + [2]) caused significant increases in Nluc activities when transfected into HEK293T cells with the 6xISRE-NlucP plasmid. After 36 h, Nluc luciferase activities were measured (mean ± SD of three independent experiments). (D) Different numbers of ISRE were placed in the promoter of NlucP (SI Appendix, Fig. S1B) and transfected into HEK293T cells in a 96-well plate (Negative: [3] + [4] and Positive: [3] + [2]). After 36 h, the Nluc assay was performed. Asterisks indicate significant difference compared each Positive to Negative (*P < 0.01, **P < 0.0001). IFNAR1 and IFNAR2, interferon alpha, and beta receptor subunits 1 and 2; EX, extracellular domain; TM, transmembrane region; IN, intracellular domain; JAK, Janus kinase; TYK2, tyrosine kinase 2; STAT, signal transducer and activator of transcription; IRF9, interferon regulatory factor 9; ISRE, interferon-stimulated response element; NlucP, a destabilized form of Nluc with a PEST sequence; PM, plasma membrane; HER2, human EGFR- related 2; and affibody, Af.

Article Snippet: The expression vectors for HER2 (#16257), CD8α- EGFP (#86051), TCRα/β/CD3ε/ζ (#89347), Cas9 (#52961), pSLCAR- CD1928z (#135991), and pSLCAR- CD19- BBz (#135992) were obtained from Addgene.

Techniques: Binding Assay, Activity Assay, Labeling, Activation Assay, Knock-Out, Transfection, Plasmid Preparation, Luciferase, Sequencing, Clinical Proteomics, Membrane

Fig. 2. IFNARRS compared with the NanoBiT system. (A–C) Signaling via the transmembrane and intracellular domains of IFNAR1/2. The same HER2-EX and anti-HER2 affibody in Fig. 1B were used in NanoBiT system (A) and IFNARRS (B). (A) The HER2-EX and anti-HER2 affibody were fused to SmBiT and LgBiT, with or without a short peptide linker (Linker) as depicted in SI Appendix, Fig. S2 (Negative: [6] + [8] or Positive: [6] + [4]). (B) The HER2-EX and anti-HER2 affibody were fused to IFNAR2-TM-IN and IFNAR1-TM-IN depicted in Fig. 1B (Negative: [3] + [4] or Positive: [3] + [2]). (C) HEK293T cells in a 96-well plate were transfected with the indicated combinations using the same backbone of the plasmid for each gene expression, using 0.2μL Lipofectamin 2000 [(A) 15 ng of -SmBiT, 15 ng of -LgBiT, and 10 ng of vector plasmids; (B) 15 ng of -IFNAR2, 15 ng of -IFNAR1, 10 ng of Nx ISRE-NlucP plasmids]. After 36 h, the Nluc assay was performed with the same amount of substrate (mean ± SD of three independent experiments). (D–F) Signaling via the intracellular domains of IFNAR1/2 alone. The same extracellular and transmembrane regions of HER2 (HER2-EX-TM) in SI Appendix, Fig. S5A were used in NanoBiT system (D) and IFNARRS (E). (D) The HER2-EX- TM was fused to SmBiT and LgBiT with or without Linker, as depicted in SI Appendix, Fig. S6. [2] HER2-EX-TM-Linker-SmBiT and [4] HER2-EX-TM-Linker-LgBiT successfully generated the anti-HER2 affibody dimer-dependent signal (Negative: vector or Positive: anti-HER2 Af). (E) The HER2-EX-TM was fused to the IFNAR1 and IFNAR2 intracellular domains (IFNAR1-IN and IFNAR2-IN). As shown in SI Appendix, Fig. S5, the intracellular [1] IFNAR2-IN-265 and [6] IFNAR1-IN-D62 domains successfully transmitted the signal of the JAK-STAT pathway (Negative: vector or Positive: anti-HER2 Af). (F) To compare this IFNARRS to the NanoBiT system for the anti-HER2 affibody dimer-dependent signal, HEK293T cells in a 96-well plate were transfected with the indicated plasmids, using 0.2 μL Lipofectamin 2000 [(D) 10 ng of -SmBiT, 10 ng of -LgBiT, and 10 ng of vector plasmids with 10 ng of vector or anti-HER2 Af plasmid; (E) 10 ng of -IFNAR2, 10 ng of -IFNAR1, and 10 ng of Nx ISRE-NlucP plasmids with 10 ng of vector or anti-HER2 Af plasmid]. After 36 h, Nluc assay was performed with the same amount of substrate (mean ± SD of three independent experiments). Asterisks indicate significant difference compared each Positive to Negative (*P < 0.01, **P < 0.0001).

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Development of a highly sensitive platform for protein-protein interaction detection and regulation of T cell function.

doi: 10.1073/pnas.2318190121

Figure Lengend Snippet: Fig. 2. IFNARRS compared with the NanoBiT system. (A–C) Signaling via the transmembrane and intracellular domains of IFNAR1/2. The same HER2-EX and anti-HER2 affibody in Fig. 1B were used in NanoBiT system (A) and IFNARRS (B). (A) The HER2-EX and anti-HER2 affibody were fused to SmBiT and LgBiT, with or without a short peptide linker (Linker) as depicted in SI Appendix, Fig. S2 (Negative: [6] + [8] or Positive: [6] + [4]). (B) The HER2-EX and anti-HER2 affibody were fused to IFNAR2-TM-IN and IFNAR1-TM-IN depicted in Fig. 1B (Negative: [3] + [4] or Positive: [3] + [2]). (C) HEK293T cells in a 96-well plate were transfected with the indicated combinations using the same backbone of the plasmid for each gene expression, using 0.2μL Lipofectamin 2000 [(A) 15 ng of -SmBiT, 15 ng of -LgBiT, and 10 ng of vector plasmids; (B) 15 ng of -IFNAR2, 15 ng of -IFNAR1, 10 ng of Nx ISRE-NlucP plasmids]. After 36 h, the Nluc assay was performed with the same amount of substrate (mean ± SD of three independent experiments). (D–F) Signaling via the intracellular domains of IFNAR1/2 alone. The same extracellular and transmembrane regions of HER2 (HER2-EX-TM) in SI Appendix, Fig. S5A were used in NanoBiT system (D) and IFNARRS (E). (D) The HER2-EX- TM was fused to SmBiT and LgBiT with or without Linker, as depicted in SI Appendix, Fig. S6. [2] HER2-EX-TM-Linker-SmBiT and [4] HER2-EX-TM-Linker-LgBiT successfully generated the anti-HER2 affibody dimer-dependent signal (Negative: vector or Positive: anti-HER2 Af). (E) The HER2-EX-TM was fused to the IFNAR1 and IFNAR2 intracellular domains (IFNAR1-IN and IFNAR2-IN). As shown in SI Appendix, Fig. S5, the intracellular [1] IFNAR2-IN-265 and [6] IFNAR1-IN-D62 domains successfully transmitted the signal of the JAK-STAT pathway (Negative: vector or Positive: anti-HER2 Af). (F) To compare this IFNARRS to the NanoBiT system for the anti-HER2 affibody dimer-dependent signal, HEK293T cells in a 96-well plate were transfected with the indicated plasmids, using 0.2 μL Lipofectamin 2000 [(D) 10 ng of -SmBiT, 10 ng of -LgBiT, and 10 ng of vector plasmids with 10 ng of vector or anti-HER2 Af plasmid; (E) 10 ng of -IFNAR2, 10 ng of -IFNAR1, and 10 ng of Nx ISRE-NlucP plasmids with 10 ng of vector or anti-HER2 Af plasmid]. After 36 h, Nluc assay was performed with the same amount of substrate (mean ± SD of three independent experiments). Asterisks indicate significant difference compared each Positive to Negative (*P < 0.01, **P < 0.0001).

Techniques: Transfection, Plasmid Preparation, Gene Expression, Generated

Fig. 7. Expressions of B7-1, anti-CD3ε-scFv-TM, and ZZ-TM in the GRkS-974, GRkSB-043, and GRkSBZ-188 cells. (A) To confirm IFNγ-dependent expression of B7-1 in GRkSB-043 and GRkSBZ-188 cells, the GRkS-974, GRkSB-043, and GRkSBZ-188 cells (1 × 105, each) on a 24-well plate were treated with 1ng/mL IFNγ for 24 h. The cell lysate (5 μg protein of each) was dotted onto a nitrocellulose membrane. The B7-1 proteins were visualized with an anti-B7-1 antibody, HRP- conjugated anti-mouse IgG antibody, and ECL reagent. (B). GRkS-974, GRkSB-043, and GRkSBZ-188 cells (1 × 105 each) on a 24-well plate were treated with 1ng/ mL IFNγ for 24 h. The 1 μg of total RNA was subjected to reverse transcription and used in PCR using the specific primers (for anti-CD3ε-scFv-TM). The products were electrophoresed in a 1% agarose gel and stained with ethidium bromide. The arrow indicates the expected PCR product (0.35 kb). (C) and (D) show specific bindings of NlucP-IGHG1 and anti-HER2-affibody-IGHG1 to the surface ZZ-TM on GRkSBZ-188 cells. In C, plasmids designed to secrete NlucP or NlucP-fused IGHG1 proteins were transfected to HEK293T cells, the supernatants were collected, and one aliquot was subjected to binding assay. The same counts (25,000) of NlucP or NlucP-IGHG1 were incubated with GRkS-974, GRkSB-043, and GRkSBZ-188 cells for 12 h. After removing the supernatant, the Nluc activities bound to cells were measured and shown as Bound/Input. Compared to NlucP, NlucP-IGHG1 was bound specifically to GRkSBZ-188 cells. In D, plasmids designed to secrete HER2-EX-NlucP or anti-HER2-affibody-IGHG1 proteins were transfected to HEK293T cells (1 × 105 each), the supernatants were collected, and one aliquot was subjected to binding assay. The same counts (15,000) of HER2-EX-NlucP with or without anti-HER2-affibody-IGHG1 were incubated with GRkS-974, GRkSB-043, and GRkSBZ-188 cells for 12 h. After removing the supernatant, the Nluc activities bound to cells were measured and shown as Bound/Input. Compared to HER2-EX-NlucP only, HER2-EX-NlucP with anti-HER2-affibody-IGHG1 specifically bound to GRkSBZ-188 cells. Asterisks indicate a significant difference from the control (C, NlucP or D, HER2-EX-NlucP only) (*P < 0.0001, **P < 0.000001).

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Development of a highly sensitive platform for protein-protein interaction detection and regulation of T cell function.

doi: 10.1073/pnas.2318190121

Figure Lengend Snippet: Fig. 7. Expressions of B7-1, anti-CD3ε-scFv-TM, and ZZ-TM in the GRkS-974, GRkSB-043, and GRkSBZ-188 cells. (A) To confirm IFNγ-dependent expression of B7-1 in GRkSB-043 and GRkSBZ-188 cells, the GRkS-974, GRkSB-043, and GRkSBZ-188 cells (1 × 105, each) on a 24-well plate were treated with 1ng/mL IFNγ for 24 h. The cell lysate (5 μg protein of each) was dotted onto a nitrocellulose membrane. The B7-1 proteins were visualized with an anti-B7-1 antibody, HRP- conjugated anti-mouse IgG antibody, and ECL reagent. (B). GRkS-974, GRkSB-043, and GRkSBZ-188 cells (1 × 105 each) on a 24-well plate were treated with 1ng/ mL IFNγ for 24 h. The 1 μg of total RNA was subjected to reverse transcription and used in PCR using the specific primers (for anti-CD3ε-scFv-TM). The products were electrophoresed in a 1% agarose gel and stained with ethidium bromide. The arrow indicates the expected PCR product (0.35 kb). (C) and (D) show specific bindings of NlucP-IGHG1 and anti-HER2-affibody-IGHG1 to the surface ZZ-TM on GRkSBZ-188 cells. In C, plasmids designed to secrete NlucP or NlucP-fused IGHG1 proteins were transfected to HEK293T cells, the supernatants were collected, and one aliquot was subjected to binding assay. The same counts (25,000) of NlucP or NlucP-IGHG1 were incubated with GRkS-974, GRkSB-043, and GRkSBZ-188 cells for 12 h. After removing the supernatant, the Nluc activities bound to cells were measured and shown as Bound/Input. Compared to NlucP, NlucP-IGHG1 was bound specifically to GRkSBZ-188 cells. In D, plasmids designed to secrete HER2-EX-NlucP or anti-HER2-affibody-IGHG1 proteins were transfected to HEK293T cells (1 × 105 each), the supernatants were collected, and one aliquot was subjected to binding assay. The same counts (15,000) of HER2-EX-NlucP with or without anti-HER2-affibody-IGHG1 were incubated with GRkS-974, GRkSB-043, and GRkSBZ-188 cells for 12 h. After removing the supernatant, the Nluc activities bound to cells were measured and shown as Bound/Input. Compared to HER2-EX-NlucP only, HER2-EX-NlucP with anti-HER2-affibody-IGHG1 specifically bound to GRkSBZ-188 cells. Asterisks indicate a significant difference from the control (C, NlucP or D, HER2-EX-NlucP only) (*P < 0.0001, **P < 0.000001).

Techniques: Expressing, Membrane, Reverse Transcription, Agarose Gel Electrophoresis, Staining, Transfection, Binding Assay, Incubation, Control

Exosomes derived from MDA-MB-231 cells stimulate RANKL-induced osteoclastogenesis in vitro . (A) Representative transmission electron microscopy images of exosomes derived from MDA-MB-231 cells. Scale bar, 500 nm (left panel) and 100 nm (right panel). (B) Nanoparticle tracking analysis of exosomes derived from MDA-MB-231 cells. (C) Western blot analysis of whole cell lysates (WC) and exosomes (EXOs) prepared from MDA-MB-231 cells. (D) BMMs were treated with exosomes derived from MDA-MB-231 cells in the presence of RANKL and M-CSF for 4 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (E) BMMs were treated with exosomes derived from MDA-MB-231 cells treated with vehicle (Con) or the pan RAS inhibitor, salirasib (10 µ M), in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. ** P<0.01. (F) BMMs were treated with exosomes derived from MCF-7 cells transfected with control vector (Con) or K-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. *** P<0.001. (G) BMMs were treated with exosomes derived from T47D cells transfected with control vector (Con), K-RASV12, H-RASV12 , or N-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (H) BMMs were treated with exosomes derived from control vector (Con) or HER2 vector-transfected T47D cells in the presence of RANKL and M-CSF for 6 days. * P<0.05 and **** P<0.0001. RANKL, receptor activator of nuclear factor-κB ligand; BMMs, bone marrow-derived macrophages; TRAP, tartrate-resistant acid phosphatase; M-CSF, macrophage colony-stimulating factor; EXOs, exosomes; ALIX, apoptosis-linked gene 2-interacting protein X; HSP, heat shock protein; TSG101, tumor susceptibility 101; GM130, Golgi matrix protein 130; HER2, human epidermal growth factor receptor 2.

Journal: International Journal of Molecular Medicine

Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells

doi: 10.3892/ijmm.2023.5287

Figure Lengend Snippet: Exosomes derived from MDA-MB-231 cells stimulate RANKL-induced osteoclastogenesis in vitro . (A) Representative transmission electron microscopy images of exosomes derived from MDA-MB-231 cells. Scale bar, 500 nm (left panel) and 100 nm (right panel). (B) Nanoparticle tracking analysis of exosomes derived from MDA-MB-231 cells. (C) Western blot analysis of whole cell lysates (WC) and exosomes (EXOs) prepared from MDA-MB-231 cells. (D) BMMs were treated with exosomes derived from MDA-MB-231 cells in the presence of RANKL and M-CSF for 4 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (E) BMMs were treated with exosomes derived from MDA-MB-231 cells treated with vehicle (Con) or the pan RAS inhibitor, salirasib (10 µ M), in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. ** P<0.01. (F) BMMs were treated with exosomes derived from MCF-7 cells transfected with control vector (Con) or K-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. *** P<0.001. (G) BMMs were treated with exosomes derived from T47D cells transfected with control vector (Con), K-RASV12, H-RASV12 , or N-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (H) BMMs were treated with exosomes derived from control vector (Con) or HER2 vector-transfected T47D cells in the presence of RANKL and M-CSF for 6 days. * P<0.05 and **** P<0.0001. RANKL, receptor activator of nuclear factor-κB ligand; BMMs, bone marrow-derived macrophages; TRAP, tartrate-resistant acid phosphatase; M-CSF, macrophage colony-stimulating factor; EXOs, exosomes; ALIX, apoptosis-linked gene 2-interacting protein X; HSP, heat shock protein; TSG101, tumor susceptibility 101; GM130, Golgi matrix protein 130; HER2, human epidermal growth factor receptor 2.

Article Snippet: The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological.

Techniques: Derivative Assay, In Vitro, Transmission Assay, Electron Microscopy, Western Blot, Staining, Transfection, Plasmid Preparation

Identification of osteoclastogenic miRNAs in exosomes induced by RAS activation. (A) Expression levels of 28 miRNAs in exosomes derived from MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. (B) The expression levels of 11 selected miRNAs in exosomes derived from control or KRASV12 vector-transfected MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (C) Cellular expression levels of 11 selected miRNAs in MCF-7 cells transfected with the control or K-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. (D) The expression levels of eight selected miRNAs in exosomes derived from MDA-MB-231 cells treated with the control or salirasib (10 µ M) were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (E) The expression levels of seven selected miRNAs in exosomes derived from T47D cells transfected with the control vector (Con) or N-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05, ** P<0.01 and **** P<0.0001. (F) Representative images of TRAP-positive osteoclasts in BMMs transfected with the indicated miRNAs. BMMs transfected with the indicated miRNAs (20 nM each) were stimulated with RANKL and M-CSF for 4 days. NC, miRNA mimic negative control. ** P<0.01. (G) The expression levels of miR-494-3p, miR-1915-3p, miR-4508 and miR-6869-5p in sera derived from patients with HER2-positive breast cancer (n=19) and triple-negative breast cancer (n=15). The results were normalized to U6 snRNA. * P<0.05, *** P<0.001 and **** P<0.0001. RT-qPCR, reverse transcription-quantitative PCR; M-CSF, macrophage colony-stimulating factor; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer; BMMs, bone marrow-derived macrophages.

Journal: International Journal of Molecular Medicine

Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells

doi: 10.3892/ijmm.2023.5287

Figure Lengend Snippet: Identification of osteoclastogenic miRNAs in exosomes induced by RAS activation. (A) Expression levels of 28 miRNAs in exosomes derived from MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. (B) The expression levels of 11 selected miRNAs in exosomes derived from control or KRASV12 vector-transfected MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (C) Cellular expression levels of 11 selected miRNAs in MCF-7 cells transfected with the control or K-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. (D) The expression levels of eight selected miRNAs in exosomes derived from MDA-MB-231 cells treated with the control or salirasib (10 µ M) were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (E) The expression levels of seven selected miRNAs in exosomes derived from T47D cells transfected with the control vector (Con) or N-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05, ** P<0.01 and **** P<0.0001. (F) Representative images of TRAP-positive osteoclasts in BMMs transfected with the indicated miRNAs. BMMs transfected with the indicated miRNAs (20 nM each) were stimulated with RANKL and M-CSF for 4 days. NC, miRNA mimic negative control. ** P<0.01. (G) The expression levels of miR-494-3p, miR-1915-3p, miR-4508 and miR-6869-5p in sera derived from patients with HER2-positive breast cancer (n=19) and triple-negative breast cancer (n=15). The results were normalized to U6 snRNA. * P<0.05, *** P<0.001 and **** P<0.0001. RT-qPCR, reverse transcription-quantitative PCR; M-CSF, macrophage colony-stimulating factor; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer; BMMs, bone marrow-derived macrophages.

Article Snippet: The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological.

Techniques: Activation Assay, Expressing, Derivative Assay, Quantitative RT-PCR, Plasmid Preparation, Transfection, Negative Control, Real-time Polymerase Chain Reaction

( a ) Pattern and frequency of MAPK pathway activating mutations (OncoKb annotated) in 733 ERBB2-amplified breast tumors, stratified based on tumor type. ( b ) Frequency of MAPK alterations from ( a ) in metastatic and primary tumor samples, p < 0.05 by two-sided Fisher exact test. ( c ) Kaplan-Meier curve displaying progression-free survival of patients receiving first-line anti-HER2 therapy. Analysis was restricted to patients for whom genomic profiling was performed on a tumor specimen prior to starting first-line therapy, n = 145. Tumors with functional alterations in MAPK signaling members are shown in green, and tumors without MAPK alterations are shown in blue. P < 0.05, two-sided log-rank test. ( d ) The emergence of an NF1- loss of function alteration after exposure to anti-HER2 targeted therapy. This is a case of a 38-year-old female patient with de novo metastatic HER2-positive invasive ductal carcinoma of the left breast, who received first-line treatment with docetaxel, trastuzumab, and pertuzumab (THP), followed by maintenance with trastuzumab and pertuzumab (HP). She experienced a partial response (PR) after 6 months of therapy, which was maintained on HP for 31.5 months (959 days) when she experienced an isolated progression on the left axillary lymph nodes. Fused PET/CT scan axial images of PET Scan on HP and at the time of progression have been shown. Samples of the primary breast tumor and samples of progressing axillary lymph nodes, collected before and after exposure to anti-HER2 therapy, have been sequenced. ( e ) An NF1intragenic inversion (c.1527 + 970:NF1_chr17:g.57371683inv) was detected on left axillary lymph node biopsy but not in the pre-treatment tumor. This rearrangement extends 27 megabases, bisecting NF1 and resulting in inversion of exons 14-58 of NF1; this is predicted to result in loss of function due to involvement and complete inversion of the RAS GTPase domain. Abbreviations: IDC: invasive ductal carcinoma; ER: estrogen receptor: PR: progesterone receptor; POD: progression of disease; PR: partial response; PET: positron emission tomography; CT: computed tomography.

Journal: Nature Communications

Article Title: HER2 + breast cancers evade anti-HER2 therapy via a switch in driver pathway

doi: 10.1038/s41467-021-27093-y

Figure Lengend Snippet: ( a ) Pattern and frequency of MAPK pathway activating mutations (OncoKb annotated) in 733 ERBB2-amplified breast tumors, stratified based on tumor type. ( b ) Frequency of MAPK alterations from ( a ) in metastatic and primary tumor samples, p < 0.05 by two-sided Fisher exact test. ( c ) Kaplan-Meier curve displaying progression-free survival of patients receiving first-line anti-HER2 therapy. Analysis was restricted to patients for whom genomic profiling was performed on a tumor specimen prior to starting first-line therapy, n = 145. Tumors with functional alterations in MAPK signaling members are shown in green, and tumors without MAPK alterations are shown in blue. P < 0.05, two-sided log-rank test. ( d ) The emergence of an NF1- loss of function alteration after exposure to anti-HER2 targeted therapy. This is a case of a 38-year-old female patient with de novo metastatic HER2-positive invasive ductal carcinoma of the left breast, who received first-line treatment with docetaxel, trastuzumab, and pertuzumab (THP), followed by maintenance with trastuzumab and pertuzumab (HP). She experienced a partial response (PR) after 6 months of therapy, which was maintained on HP for 31.5 months (959 days) when she experienced an isolated progression on the left axillary lymph nodes. Fused PET/CT scan axial images of PET Scan on HP and at the time of progression have been shown. Samples of the primary breast tumor and samples of progressing axillary lymph nodes, collected before and after exposure to anti-HER2 therapy, have been sequenced. ( e ) An NF1intragenic inversion (c.1527 + 970:NF1_chr17:g.57371683inv) was detected on left axillary lymph node biopsy but not in the pre-treatment tumor. This rearrangement extends 27 megabases, bisecting NF1 and resulting in inversion of exons 14-58 of NF1; this is predicted to result in loss of function due to involvement and complete inversion of the RAS GTPase domain. Abbreviations: IDC: invasive ductal carcinoma; ER: estrogen receptor: PR: progesterone receptor; POD: progression of disease; PR: partial response; PET: positron emission tomography; CT: computed tomography.

Article Snippet: Stable overexpression of cyclin E2 was achieved by cloning the insert from pcDNA3-HA-cyclin E2 (addgene #19935) into pLenti CMV Puro DEST (addgene #17452). pLX302 and pLX302-HER2 L755S expression vectors were gifts from Maurizio Scaltriti.

Techniques: Amplification, Functional Assay, Isolation, Positron Emission Tomography-Computed Tomography, Positron Emission Tomography, Computed Tomography

( a ) Proliferation of shRenilla control and shNF1 HER2 + breast cancer cell lines exposed to 500 nM (SKBR3, MDA-MB-361, BT-474) or 2 uM (HCC1954) lapatinib. shNF1 #1 and #2 represent unique short hairpin sequences targeting NF1. Data are means + /- SD of six biological replicates. ( b ) Proliferation of shRenilla and shNF1 expressing SKBR3 cells exposed to 50 nM neratinib or 100 nM tucatinib. Data are means of 6 biological replicates ± SD. ( c ) Crystal violet staining of shRenilla and shNF1 SKBR3 cells exposed to 500 nM lapatinib (HER2i) over 30 days. ( d ) Immunoblots of indicated hosphor ( p ) and total proteins in shRen and shNF1 SKBR3 cells were treated with 500 nM lapatinib and collected at specified times. Densitometric quantification values are provided below immunoblots. Phospho-signal intensities were normalized to respective total protein signals. Data are representative of 3 biological replicates. ( e ) Proliferation of lapatinib resistant (HER2i-R) shNF1 SKBR3 cells transduced with a doxycycline (dox)-inducible NF1 or empty control vector, treated with dox ± 500 nM lapatinib. Data are means of 6 biological replicates, ± SD. ( f ) Proliferation of SKBR3 cells transduced with vectors constitutively expressing HER2 L755S, BRAF V600E, KRAS G12V, or shRNAs against NF1 continuously treated with 500 nM lapatinib. Data points are means ± SD, n = 6 biological replicates. Source data for all assays are provided as a Source Data file.

Journal: Nature Communications

Article Title: HER2 + breast cancers evade anti-HER2 therapy via a switch in driver pathway

doi: 10.1038/s41467-021-27093-y

Figure Lengend Snippet: ( a ) Proliferation of shRenilla control and shNF1 HER2 + breast cancer cell lines exposed to 500 nM (SKBR3, MDA-MB-361, BT-474) or 2 uM (HCC1954) lapatinib. shNF1 #1 and #2 represent unique short hairpin sequences targeting NF1. Data are means + /- SD of six biological replicates. ( b ) Proliferation of shRenilla and shNF1 expressing SKBR3 cells exposed to 50 nM neratinib or 100 nM tucatinib. Data are means of 6 biological replicates ± SD. ( c ) Crystal violet staining of shRenilla and shNF1 SKBR3 cells exposed to 500 nM lapatinib (HER2i) over 30 days. ( d ) Immunoblots of indicated hosphor ( p ) and total proteins in shRen and shNF1 SKBR3 cells were treated with 500 nM lapatinib and collected at specified times. Densitometric quantification values are provided below immunoblots. Phospho-signal intensities were normalized to respective total protein signals. Data are representative of 3 biological replicates. ( e ) Proliferation of lapatinib resistant (HER2i-R) shNF1 SKBR3 cells transduced with a doxycycline (dox)-inducible NF1 or empty control vector, treated with dox ± 500 nM lapatinib. Data are means of 6 biological replicates, ± SD. ( f ) Proliferation of SKBR3 cells transduced with vectors constitutively expressing HER2 L755S, BRAF V600E, KRAS G12V, or shRNAs against NF1 continuously treated with 500 nM lapatinib. Data points are means ± SD, n = 6 biological replicates. Source data for all assays are provided as a Source Data file.

Techniques: Expressing, Staining, Western Blot, Transduction, Plasmid Preparation

( a ) Inhibition of proliferation of SKBR3 shRenilla and shNF1 HER2i-R cells by lapatinib (HER2i), MK2206 (AKTi), trametinib (MEKi), and SCH772984 (ERKi), plotted as % inhibition of proliferation after 5 days treatment vs log concentration of drug (nM). Data represent means of 6 biological replicates. ( b ) Crystal violet staining of shRenilla and shNF1 HER2i-R SKBR3 cells exposed to indicated doses of trametinib (MEKi) over 7 days. Data are representative images of 3 biological replicates. ( c ) Inhibition of proliferation of shRen and HER2 inhibitor resistant (HER2i-R) shNF1, HER2 L755S-expressing, and KRAS G12V-expressing SKBR3 cells by trametinib (MEKi). ( d ) Growth of BT-474 shRenilla and shNF1 HER2i-R xenograft tumors treated with vehicle or 1 mg/kg trametinib daily. n = 10 mice per group, data are means ± SEM. P value = 0.0059 by two-sided student’s t -test. ( e ) Immunohistochemistry staining of phosphor-ERK1/2 in BT-474 xenograft tumor sections from ( d ). Images are representative of 4 tumors per treatment group. Scale bars = 100 um ( f ) Growth of patient-derived xenograft harboring ERBB2 amplification and NF1 deletion in mice treated with vehicle, 10 mg/kg trastuzumab bi-weekly, 1 mg/kg trametinib weekly, or the combination. N = 5 mice per group bearing two tumors each, data are means ± SEM. P value < 0.00103 by two-sided student’s t -test. ( g ) Immunohistochemistry staining of phospho-ERK in NF1 null PDX tumors from ( f ), scale bar 100 um. Source data for all assays are provided as a Source Data file.

Journal: Nature Communications

Article Title: HER2 + breast cancers evade anti-HER2 therapy via a switch in driver pathway

doi: 10.1038/s41467-021-27093-y

Figure Lengend Snippet: ( a ) Inhibition of proliferation of SKBR3 shRenilla and shNF1 HER2i-R cells by lapatinib (HER2i), MK2206 (AKTi), trametinib (MEKi), and SCH772984 (ERKi), plotted as % inhibition of proliferation after 5 days treatment vs log concentration of drug (nM). Data represent means of 6 biological replicates. ( b ) Crystal violet staining of shRenilla and shNF1 HER2i-R SKBR3 cells exposed to indicated doses of trametinib (MEKi) over 7 days. Data are representative images of 3 biological replicates. ( c ) Inhibition of proliferation of shRen and HER2 inhibitor resistant (HER2i-R) shNF1, HER2 L755S-expressing, and KRAS G12V-expressing SKBR3 cells by trametinib (MEKi). ( d ) Growth of BT-474 shRenilla and shNF1 HER2i-R xenograft tumors treated with vehicle or 1 mg/kg trametinib daily. n = 10 mice per group, data are means ± SEM. P value = 0.0059 by two-sided student’s t -test. ( e ) Immunohistochemistry staining of phosphor-ERK1/2 in BT-474 xenograft tumor sections from ( d ). Images are representative of 4 tumors per treatment group. Scale bars = 100 um ( f ) Growth of patient-derived xenograft harboring ERBB2 amplification and NF1 deletion in mice treated with vehicle, 10 mg/kg trastuzumab bi-weekly, 1 mg/kg trametinib weekly, or the combination. N = 5 mice per group bearing two tumors each, data are means ± SEM. P value < 0.00103 by two-sided student’s t -test. ( g ) Immunohistochemistry staining of phospho-ERK in NF1 null PDX tumors from ( f ), scale bar 100 um. Source data for all assays are provided as a Source Data file.

Techniques: Inhibition, Concentration Assay, Staining, Expressing, Immunohistochemistry, Derivative Assay, Amplification

HER2-PI9 and HER2-I12 novel splice variant transcript identification. Conventional PCR was used to screen for alternative splice variants of HER2 in the SKBR3 (HER2 +) cell line. Primers used included; HER2 exon 8 forward 5'-AACACAGCGGTGTGAGAAGT-3'; HER2 exon 10 reverse 5'-GTGATCTCTTCCAGAGTCTC-3'; HER2 exon 12 forward 5'-GGCCAGAGGACGAGTGTG-3', HER2 exon 14 reverse 5'-CGGTCCAAAACAGGTCACT-3'; β Actin forward 5'-GGACTTCGAGCAAGAGATGG-3'; β Actin reverse 5'-AGCACTGTGTTGGCGTACAG-3'. A Alternative splicing produces a variant transcript with partial intron 9 inclusion. The HER2-PI9 variant transcript includes a 117 bp cassette-exon sequence (underlined). B Intron 12 (underlined) is retained by alternative splicing to produce the HER2-I12 variant transcript

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: HER2-PI9 and HER2-I12 novel splice variant transcript identification. Conventional PCR was used to screen for alternative splice variants of HER2 in the SKBR3 (HER2 +) cell line. Primers used included; HER2 exon 8 forward 5'-AACACAGCGGTGTGAGAAGT-3'; HER2 exon 10 reverse 5'-GTGATCTCTTCCAGAGTCTC-3'; HER2 exon 12 forward 5'-GGCCAGAGGACGAGTGTG-3', HER2 exon 14 reverse 5'-CGGTCCAAAACAGGTCACT-3'; β Actin forward 5'-GGACTTCGAGCAAGAGATGG-3'; β Actin reverse 5'-AGCACTGTGTTGGCGTACAG-3'. A Alternative splicing produces a variant transcript with partial intron 9 inclusion. The HER2-PI9 variant transcript includes a 117 bp cassette-exon sequence (underlined). B Intron 12 (underlined) is retained by alternative splicing to produce the HER2-I12 variant transcript

Article Snippet: Expression vectors were produced using a HER2 expression vector, a gift from Mien-Chie Hung (Addgene plasmid #16,257; http://n2t.net/addgene:16257 ; RRID:Addgene_16257, Massachusetts, USA).

Techniques: Variant Assay, Alternative Splicing, Sequencing

Detection of HER2-PI9 and HER2-I12 splice variants in breast cancer cell lines and tissues. A The relative mRNA expression of HER2-WT, HER2-PI9 and HER2-I12 in MCF-7 (HER2-), T47D (HER2 −), MDA-MB-231 (HER2 −) and SKBR3 (HER2 +) breast cancer cell lines. Taqman real-time PRC assays were designed and validated to correspond to each splice variant. HER2-WT was significantly higher in the HER2 + cell line SKBR3, compared to all the HER2- lines including MCF-7 ( p < 0.0001), T47D ( p < 0.0001) and MDA-MB-231 ( p = 0.0002). HER2-PI9 had particularly high relative expression in the cell line MDA-MB-231, compared to the cell lines MCF-7, ( p < 0.0001), T47D ( p = 0.0002) and SKBR3 ( p < 0.0001). HER-I12 expression was significantly higher in T47D compared to MCF-7 ( p = 0.0247) and MDA-MB-231 compared to MCF-7 ( p < 0.0001) or SKBR3 ( p < 0.0001). The line MDA-MB-231 expresses HER-I12 to a greater extent than T47D ( p < 0.0001). B HER2-WT, HER2-PI9 and HER2-I12 mRNA relative expression in human tissues. The HER2 splice variants are all present in cerebellum, heart, liver, muscle, testis and normal breast tissues although no tissue had significantly higher expression than another. Data is normalized to the housekeeper β-actin. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, one-way ANOVA

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Detection of HER2-PI9 and HER2-I12 splice variants in breast cancer cell lines and tissues. A The relative mRNA expression of HER2-WT, HER2-PI9 and HER2-I12 in MCF-7 (HER2-), T47D (HER2 −), MDA-MB-231 (HER2 −) and SKBR3 (HER2 +) breast cancer cell lines. Taqman real-time PRC assays were designed and validated to correspond to each splice variant. HER2-WT was significantly higher in the HER2 + cell line SKBR3, compared to all the HER2- lines including MCF-7 ( p < 0.0001), T47D ( p < 0.0001) and MDA-MB-231 ( p = 0.0002). HER2-PI9 had particularly high relative expression in the cell line MDA-MB-231, compared to the cell lines MCF-7, ( p < 0.0001), T47D ( p = 0.0002) and SKBR3 ( p < 0.0001). HER-I12 expression was significantly higher in T47D compared to MCF-7 ( p = 0.0247) and MDA-MB-231 compared to MCF-7 ( p < 0.0001) or SKBR3 ( p < 0.0001). The line MDA-MB-231 expresses HER-I12 to a greater extent than T47D ( p < 0.0001). B HER2-WT, HER2-PI9 and HER2-I12 mRNA relative expression in human tissues. The HER2 splice variants are all present in cerebellum, heart, liver, muscle, testis and normal breast tissues although no tissue had significantly higher expression than another. Data is normalized to the housekeeper β-actin. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, one-way ANOVA

Techniques: Expressing, Variant Assay

Expression of HER2-PI9 and HER2-I12 splice variants in invasive breast carcinomas. HER2-WT, HER2-PI9 and HER2-I12 variants were identified via RT qPCR in HER2 + ( n = 10) and HER2 − ( n = 10) invasive ductal breast carcinomas as well as in normal breast tissue ( n = 4). Black circles are ER- and blue are ER + . HER2-WT expression was significantly higher in HER2 + tumours compared to HER2 − tumours ( p = 0.0014). HER2-PI9 was present in HER2 + , HER2 − and normal breast samples. Higher expression was identified in the HER2 + groups compared to the HER2 − group ( p = 0.0259). HER2-I12 was also present in all groups and was significantly higher in the HER2 + groups compared to HER2 − ( p = 0.0009) and normal breast ( p = 0126). ER status did not significantly correlate with variant expression. Data is normalized to the housekeeper β-actin. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, one-way ANOVA

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Expression of HER2-PI9 and HER2-I12 splice variants in invasive breast carcinomas. HER2-WT, HER2-PI9 and HER2-I12 variants were identified via RT qPCR in HER2 + ( n = 10) and HER2 − ( n = 10) invasive ductal breast carcinomas as well as in normal breast tissue ( n = 4). Black circles are ER- and blue are ER + . HER2-WT expression was significantly higher in HER2 + tumours compared to HER2 − tumours ( p = 0.0014). HER2-PI9 was present in HER2 + , HER2 − and normal breast samples. Higher expression was identified in the HER2 + groups compared to the HER2 − group ( p = 0.0259). HER2-I12 was also present in all groups and was significantly higher in the HER2 + groups compared to HER2 − ( p = 0.0009) and normal breast ( p = 0126). ER status did not significantly correlate with variant expression. Data is normalized to the housekeeper β-actin. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, one-way ANOVA

Techniques: Expressing, Quantitative RT-PCR, Variant Assay

HER2-PI9 and HER2-I12 predicted protein structures and in-vitro model production. In silico predictions of protein structure for the novel splice variants based off their RNA sequence using the Expasy Translate Tool software. A HER2-WT protein structure showing the LI, LII, CI and CII extracellular domains and the intracellular tyrosine kinase (TK) domain. NH 2 amino-terminus. COOH carboxyl-terminus. B HER2-PI9 predicted protein structure including the GVQW domain (highlighted red). C HER2-I12 predicted protein structure including the novel C-terminal region (highlighted red). D Detection by western blotting of HER2-WT, HER2-PI9 and HER2-I12 proteins in MCF-7 cells transformed with expression vectors. HER2-WT and HER2-PI9 are present above the 170 kDa protein marker, correlating to the 185 kDa and 190 kDa predicted proteins respectively. HER2-I12 has a band just below the 70 kDa marker, correlating to the predicted 64 kDa protein. The empty vector control has protein expression relative to the MCF-7 control. The cell line, SKBR3, was included as a positive control as it expresses high levels of HER2-WT. 30 μg total protein was loaded in each lane. Alpha-tubulin (50 kDa) was blotted for as a loading control

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: HER2-PI9 and HER2-I12 predicted protein structures and in-vitro model production. In silico predictions of protein structure for the novel splice variants based off their RNA sequence using the Expasy Translate Tool software. A HER2-WT protein structure showing the LI, LII, CI and CII extracellular domains and the intracellular tyrosine kinase (TK) domain. NH 2 amino-terminus. COOH carboxyl-terminus. B HER2-PI9 predicted protein structure including the GVQW domain (highlighted red). C HER2-I12 predicted protein structure including the novel C-terminal region (highlighted red). D Detection by western blotting of HER2-WT, HER2-PI9 and HER2-I12 proteins in MCF-7 cells transformed with expression vectors. HER2-WT and HER2-PI9 are present above the 170 kDa protein marker, correlating to the 185 kDa and 190 kDa predicted proteins respectively. HER2-I12 has a band just below the 70 kDa marker, correlating to the predicted 64 kDa protein. The empty vector control has protein expression relative to the MCF-7 control. The cell line, SKBR3, was included as a positive control as it expresses high levels of HER2-WT. 30 μg total protein was loaded in each lane. Alpha-tubulin (50 kDa) was blotted for as a loading control

Techniques: In Vitro, In Silico, Sequencing, Software, Western Blot, Transformation Assay, Expressing, Marker, Plasmid Preparation, Control, Positive Control

$Expression of HER2-PI9 and HER2-I12 variants in cellular fractions. Western blot analysis of protein extracted from the cell growth media (extracellular), cytoplasm, membrane and nucleus. A Protein levels of the empty vector (EV) control and HER2-WT. For the EV control no HER2 protein was identified. HER2-WT (185kDA) was only expressed in the membranous fraction. B Protein levels of HER2-PI9 and HER2-I12. HER2-PI9 (190 kDa) is expressed in the membranous and nuclear fractions. HER2-I12 (64 kDa) is expressed in the membranous and nuclear fractions. 30 μg total protein was loaded in each lane. Multiple loading controls were included with each specifically expressed in a single fraction to ensure protein was only extracted from the intended cellular compartment. Loading controls included transferrin (77 kDa) (extracellular marker), α-tubulin (50 kDa) (cytoplasmic marker), pan-cadherin (100 kDa) (membrane marker) and c-jun (36 kDa) (nuclear marker) as loading controls. This experiment was run thrice and a representative image shown$

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Expression of HER2-PI9 and HER2-I12 variants in cellular fractions. Western blot analysis of protein extracted from the cell growth media (extracellular), cytoplasm, membrane and nucleus. A Protein levels of the empty vector (EV) control and HER2-WT. For the EV control no HER2 protein was identified. HER2-WT (185kDA) was only expressed in the membranous fraction. B Protein levels of HER2-PI9 and HER2-I12. HER2-PI9 (190 kDa) is expressed in the membranous and nuclear fractions. HER2-I12 (64 kDa) is expressed in the membranous and nuclear fractions. 30 μg total protein was loaded in each lane. Multiple loading controls were included with each specifically expressed in a single fraction to ensure protein was only extracted from the intended cellular compartment. Loading controls included transferrin (77 kDa) (extracellular marker), α-tubulin (50 kDa) (cytoplasmic marker), pan-cadherin (100 kDa) (membrane marker) and c-jun (36 kDa) (nuclear marker) as loading controls. This experiment was run thrice and a representative image shown

Techniques: Expressing, Western Blot, Membrane, Plasmid Preparation, Control, Marker

$Phosphorylation status of HER2-WT, HER2-PI9 and HER2-I12. Western blot analysis of segregated phosphorylated and non-phosphorylated protein extracted from cells expressing either an empty vector control (EV), HER2-WT, HER2-PI9 or HER2-I12. Protein from the MCF-7 (HER2 −) and SKBR3 (HER2 +) cell lines were included as to show weak and strong phosphorylation of HER2 proteins. A An anti-HER2 antibodies identifies strong phosphorylation of HER2 in the HER2-WT cell line which is lacking in the EV control and PI9 cell line. B HER2-I12 and SKBR3 cell lines both show strong HER2 phosphorylation. 15 μg total protein was loaded in each lane. Phosphoserine was used as a loading control to identify proteins in the phosphorylated fraction. This experiment was performed twice and a representative image shown$

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Phosphorylation status of HER2-WT, HER2-PI9 and HER2-I12. Western blot analysis of segregated phosphorylated and non-phosphorylated protein extracted from cells expressing either an empty vector control (EV), HER2-WT, HER2-PI9 or HER2-I12. Protein from the MCF-7 (HER2 −) and SKBR3 (HER2 +) cell lines were included as to show weak and strong phosphorylation of HER2 proteins. A An anti-HER2 antibodies identifies strong phosphorylation of HER2 in the HER2-WT cell line which is lacking in the EV control and PI9 cell line. B HER2-I12 and SKBR3 cell lines both show strong HER2 phosphorylation. 15 μg total protein was loaded in each lane. Phosphoserine was used as a loading control to identify proteins in the phosphorylated fraction. This experiment was performed twice and a representative image shown

Techniques: Phospho-proteomics, Western Blot, Expressing, Plasmid Preparation, Control

Effects of HER2-PI9 and HER2-I12 expression on ERK1/2 and AKT phosphorylation. Western blot analysis of the activation of the HER2 associated signalling pathways, PI3K/Akt and RAS/MAPK following transfection of MCF-7 cells with the empty vector control (EV), HER2-WT, HER2-PI9 and HER2-I12 expression vectors. A Phosphorylation of Akt (60 kDa) indicates activation of the PI3K/Akt pathway. HER2-WT or HER2-I12 expression activates Akt, shown by increased phosphorylation. B Phosphorylation of ERK 1/2 indicates activation was caused by HER2-WT or HER2-I12 expression. 30 μg total protein was loaded in each lane. Alpha-tubulin was used as a loading control (50 kDa). This experiment was run thrice and a representative image shown

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Effects of HER2-PI9 and HER2-I12 expression on ERK1/2 and AKT phosphorylation. Western blot analysis of the activation of the HER2 associated signalling pathways, PI3K/Akt and RAS/MAPK following transfection of MCF-7 cells with the empty vector control (EV), HER2-WT, HER2-PI9 and HER2-I12 expression vectors. A Phosphorylation of Akt (60 kDa) indicates activation of the PI3K/Akt pathway. HER2-WT or HER2-I12 expression activates Akt, shown by increased phosphorylation. B Phosphorylation of ERK 1/2 indicates activation was caused by HER2-WT or HER2-I12 expression. 30 μg total protein was loaded in each lane. Alpha-tubulin was used as a loading control (50 kDa). This experiment was run thrice and a representative image shown

Techniques: Expressing, Phospho-proteomics, Western Blot, Activation Assay, Transfection, Plasmid Preparation, Control

Effect of novel splice variant expression on proliferation and viability. Cell lines were produced from the HER2- cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12. A An XTT assay was used to analyse the effect of splice variant expression on cellular proliferation. B and C A trypan blue assay was also used to assess the effect of splice variant expression on proliferation and viability. The expression of HER2-WT and HER2-I12 enhanced proliferation significantly. No effect on viability was identified. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA ( n = 3)

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Effect of novel splice variant expression on proliferation and viability. Cell lines were produced from the HER2- cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12. A An XTT assay was used to analyse the effect of splice variant expression on cellular proliferation. B and C A trypan blue assay was also used to assess the effect of splice variant expression on proliferation and viability. The expression of HER2-WT and HER2-I12 enhanced proliferation significantly. No effect on viability was identified. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA ( n = 3)

Techniques: Variant Assay, Expressing, Produced, XTT Assay

Effect of novel splice variant expression on cellular migration. Cell lines were produced from the HER2 − cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12. A scratch assay was used to assess migration of the cells. Confluent cells were disrupted with a 200 μl pipette tip to produce a scratch devoid of adherent cells. At 48 h the percentage wound closure was calculated. HER2-WT and HER2-I12 expression enhanced migratory ability of the cells. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA ( n = 3)

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Effect of novel splice variant expression on cellular migration. Cell lines were produced from the HER2 − cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12. A scratch assay was used to assess migration of the cells. Confluent cells were disrupted with a 200 μl pipette tip to produce a scratch devoid of adherent cells. At 48 h the percentage wound closure was calculated. HER2-WT and HER2-I12 expression enhanced migratory ability of the cells. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA ( n = 3)

Techniques: Variant Assay, Expressing, Migration, Produced, Wound Healing Assay, Transferring

Effect of novel splice variant expression on migration. Cell lines were produced from the HER2- cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12. A boyden chamber assay was completed to assess migratory ability. The HER2 − cell line MDA-MB-231 was included as a comparison as an invasive cell line. Serum starved cells were seeded in the upper chamber and 20% foetal bovine serum-containing media was used as a chemoattractant in the lower chamber. At 24 h the cells on the transwell were fixed and stained with Mayer’s Hematoxylin Solution to assist counting. Cells that had migrated completely through the chamber were also counted. Scale bars indicate 200 μM. HER2-WT and HER2-I12 expression enhanced migratory ability of the cells. The invasive line MDA-MB-231 were also able to migrate through the boyden chamber to a greater extent than the MCF-7 cells. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA ( n = 3)

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Effect of novel splice variant expression on migration. Cell lines were produced from the HER2- cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12. A boyden chamber assay was completed to assess migratory ability. The HER2 − cell line MDA-MB-231 was included as a comparison as an invasive cell line. Serum starved cells were seeded in the upper chamber and 20% foetal bovine serum-containing media was used as a chemoattractant in the lower chamber. At 24 h the cells on the transwell were fixed and stained with Mayer’s Hematoxylin Solution to assist counting. Cells that had migrated completely through the chamber were also counted. Scale bars indicate 200 μM. HER2-WT and HER2-I12 expression enhanced migratory ability of the cells. The invasive line MDA-MB-231 were also able to migrate through the boyden chamber to a greater extent than the MCF-7 cells. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA ( n = 3)

Techniques: Variant Assay, Expressing, Migration, Produced, Boyden Chamber Assay, Comparison, Staining

Effect of novel splice variant expression on invasion. Cell lines were produced from the HER2 − cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12. A boyden chamber assay was completed to assess invasive ability. The HER2- cell line MDA-MB-231 was included as a comparison as an invasive cell line. Serum starved cells were seeded in the upper chamber atop of an extracellular matrix and 20% foetal bovine serum-containing media was used as a chemoattractant in the lower chamber. At 24 h the cells on the transwell were fixed and stained with Mayer’s Hematoxylin Solution to assist counting. Cells that had migrated completely through the chamber were also counted. Scale bars indicate 200 μM. HER2-WT and HER2-I12 expression enhanced invasive ability of the cells. The invasive line MDA-MB-231 were also able to migrate through the boyden chamber to a greater extent than the MCF-7 cells. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA ( n = 3)

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: Effect of novel splice variant expression on invasion. Cell lines were produced from the HER2 − cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12. A boyden chamber assay was completed to assess invasive ability. The HER2- cell line MDA-MB-231 was included as a comparison as an invasive cell line. Serum starved cells were seeded in the upper chamber atop of an extracellular matrix and 20% foetal bovine serum-containing media was used as a chemoattractant in the lower chamber. At 24 h the cells on the transwell were fixed and stained with Mayer’s Hematoxylin Solution to assist counting. Cells that had migrated completely through the chamber were also counted. Scale bars indicate 200 μM. HER2-WT and HER2-I12 expression enhanced invasive ability of the cells. The invasive line MDA-MB-231 were also able to migrate through the boyden chamber to a greater extent than the MCF-7 cells. Data represents mean ± SE. *** p < 0.001, ** p < 0.01, * p < 0.05, two-way ANOVA ( n = 3)

Techniques: Variant Assay, Expressing, Produced, Boyden Chamber Assay, Comparison, Staining

3D culturing of HER-WT, HER2-PI9 and HER2-I12 expressing cell lines. Cell lines were produced from the HER2 − cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12 and grown in 3D cultures. An extracellular matrix layer coated a plate to halt adherence. Cells were embedded within a 10% extracellular matrix layer with a feeding layer of complete media to ensure nutrient delivery into the system. A final cell density of 3 × 10 5 was embedded in each well. Pictures shown were taken 24 h after seeding and nine days after seeding. Scale bars indicate 200 μM

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer

doi: 10.1007/s00432-021-03689-1

Figure Lengend Snippet: 3D culturing of HER-WT, HER2-PI9 and HER2-I12 expressing cell lines. Cell lines were produced from the HER2 − cell line MCF-7, to express HER2-WT, HER2-PI9 or HER2-I12 and grown in 3D cultures. An extracellular matrix layer coated a plate to halt adherence. Cells were embedded within a 10% extracellular matrix layer with a feeding layer of complete media to ensure nutrient delivery into the system. A final cell density of 3 × 10 5 was embedded in each well. Pictures shown were taken 24 h after seeding and nine days after seeding. Scale bars indicate 200 μM

Techniques: Expressing, Produced

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human her2/erbb2 transcript variant 1 gene orf cdna clone expression plasmid (Sino Biological)

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