Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Schematic representation of the procedure steps for the preparation of LLCNs and subsequent functionalization to obtain immune-nanocapsules (LLNCs-αHER2).

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Mean diameter, standard deviation (SD), and mode of the LLNCs, LLNCs-αHER2, and LLNCs-αHER2-PC measured at 25 °C with NTA and DLS techniques in pH 7.4 buffer.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Hydrodynamic size distribution of the LLNCs, LLNCs-αHER2, and LLNCs-αHER2-PC measured at 25 °C with NTA technique in pH 7 buffer.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Physico-chemical characterization: ( a ) Zeta potential of the LLNCs (▪), LLNCs-αHER2 ( ● ), LLNCs-αHER2-FBS ( ✱ ), and LLNCs-αHER2-PC ( ▷ ) measured at 25 °C as a function of the medium pH and low ionic strength. ( b ) SDS-PAGE analysis under reducing conditions of different LLNCs. (C) Molecular weight marker: (1) αHER2; (2) FBS; (3) Fibrinogen; (4) LLNCs-αHER2; (5) elution volume after cleaning LLNCs-αHER2; (6) LLNCs-αHER2-PC; (7) elution volume after cleaning LLNCs-αHER2-PC.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: Zeta Potential Analyzer, SDS Page, Molecular Weight, Marker

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: LLNCs-αHER2-PC after incubation of immune-nanocapsules in a simulated physiological medium with FBS supplemented with FB.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: Incubation

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: Representative confocal microscopy of ( A ) SKBR3 and ( B ) HDFa (scale bar = 20 μm) incubated for 60 min with NR-LLNCs, NR-LLNCs-HER2, and NR-LLNCs-HER2-PC. Red filter and green filter correspond to LLNCs labeled with Nile Red and LLNCs labeled with HER2-FITC, respectively.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: Confocal Microscopy, Incubation, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Effect of the Protein Corona Formation on Antibody Functionalized Liquid Lipid Nanocarriers

doi: 10.3390/ijms242316759

Figure Lengend Snippet: In vitro uptake of NR-LLNCs, NR-LLNCs-HER2, and NR-LLNCs-HER2-PC in both SKBR3 and HDFa analyzed by flow cytometry. Values represent the fluorescence intensity of the merge channels for green and red as NR-LLNCs-HER2 are both red and green fluorescent. Data are mean values ± SD. * p < 0.001 shows the significant values calculated using t -test.

Article Snippet: The monoclonal ErbB-2 antibody anti-human Vio ® Bright FITC (αHER2) (clone 24D2) from humans was obtained by Miltenyi Biotech (Madrid, Spain).

Techniques: In Vitro, Flow Cytometry, Fluorescence

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: Communications biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: Fig. 1 γ-secretase inhibitor DAPT promoted differentiation of hiPSC-derived muscle progenitors. a DAPT inhibited Notch signaling by inhibiting γ- secretase. b Experimental design-1. Hu5/KD3 cells were plated onto collagen-I-coated plates and cultured for 10 days in 10% FBS/DMEM with or without DAPT, and the fusion index was determined at day 10. c Representative photos of myotube formation by Hu5/KD3 cells with or without DAPT. d Quantification of fusion index in c. Data are expressed as dot plot in control (0.1% DMSO treatment) and DAPT (10 μM DAPT treatment) cells. Data were analyzed by unpaired two-tailed Student’s t-test. n = 3 samples/group. e Quantification of myotube diameter in (c). More than 10 myotubes were measured per group. Data were analyzed by an unpaired two-tailed Student’s t-test. In d, e, the effect size of Pearson’s r correlation (r) is shown. f Experimental design 2. After 6 weeks of sphere culture-based muscle induction, cells were plated onto collagen-I-coated plates and cultured for 7 days in 10% FBS/DMEM. Then ERBB3(+)CD271(+) cells as muscle progenitors were sorted by FACS. Sorted cells were cultured for a further 6 days with (10 μM) or without DAPT (0.1% DMSO). Induction and sorting of muscle progenitors was performed twice (2 experiments per group). g Representative photos of myotubes formed by hiPSC-derived ERBB3(+)CD271(+) cells with or without DAPT. h Quantification of fusion index in g. Three wells per sample were examined. The average of each sample was shown as dot. i Distribution of myotube diameter in g. Diameter of more than 15 myotubes per sample were measured. In c and g, myotubes were stained with an antibody against skeletal muscle myosin (MF20, red) and DAPI (Nuclei, blue). Scale bars, 200 μm.

Article Snippet: The following antibodies were used in this study: CD57(HNK-1)-PE (clone TB03, Miltenyi Biotec), ERBB3-APC (clone REA508, Miltenyi Biotec), CD271-BB515 (clone C40-1457, BD Pharmingen), and human NOTCH3-PE (clone: MHN3-21, BioLegend).

Techniques: Derivative Assay, Cell Culture, Control, Two Tailed Test, Staining

Journal: Communications biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: Fig. 2 Notch inhibitor DAPT improved transplantation efficiency. a Experimental design-1. To evoke muscle regeneration, BaCl2 was injected into TA muscles of NOD-scid mice 24 h before transplantation. The next day, Hu5/KD3 cells (5.0 × 105 cells) were transplanted into damaged TA muscles with or without DAPT. TA muscles were isolated 4 weeks after transplantation. b Engraftment and differentiation of a human myoblast cell line, Hu5/KD3 cells, with or without DAPT. Donor cell-derived myofibers were detected as human lamin A/C (nuclear membrane)-positive and human spectrin (plasma membrane)-positive myofibers. Scale bar = 100 µm. c The number of human lamin A/C- and human spectrin-positive myofibers per view. Data were analyzed by unpaired two-tailed Student’s t-test. n = 3 mice/group. For each mouse, 4–8 muscle sections were examined. The effect size of Pearson’s r correlation (r) is shown. d Experimental design 2. hiPSC-derived muscle progenitors (ERBB3(+)CD271(+) cells) were transplanted into TA muscles of NSG-mdx4Cv mice with or without DAPT. BaCl2 injection and sampling of TA muscles was performed as in (a). DAPT was injected into the engrafted TA muscle four times every 3 days after transplantation. e Representative photos of engraftment and differentiation of human iPSC-derived DAPT-treated muscle progenitors. f The number of human lamin A/C- and human spectrin-positive myofibers per view. Data were analyzed by unpaired two-tailed Student’s t-test. n = 3 mice/group. For each mouse, 4–16 slices were examined. The effect size of Pearson’s r correlation (r) is also shown. In b, e, scale bar indicates 100 µm.

Article Snippet: The following antibodies were used in this study: CD57(HNK-1)-PE (clone TB03, Miltenyi Biotec), ERBB3-APC (clone REA508, Miltenyi Biotec), CD271-BB515 (clone C40-1457, BD Pharmingen), and human NOTCH3-PE (clone: MHN3-21, BioLegend).

Techniques: Transplantation Assay, Injection, Muscles, Isolation, Derivative Assay, Membrane, Clinical Proteomics, Two Tailed Test, Sampling

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet:

Article Snippet: Her2 (ERBB2) Human Tagged ORF Clone , Origene , Cat# RC222909.

Techniques: Labeling, Recombinant, Electron Microscopy, Plasmid Preparation, Blocking Assay, Software

Journal: Oncology Reports

Article Title: Erb-B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53-responsive microRNA-3184-5p in cervical cancer cells

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: ERBB2 expression is upregulated in patient-derived cervical cancer tissues and is associated with a poor prognosis. (A) RT-qPCR and (B) WB analysis of ERBB2 transcript and protein expression, respectively, in patient-derived cervical cancer tissues (n=65) vs. matched healthy cervical tissues (n=65). Data were analyzed via Wilcoxon signed-rank test. (C) RT-qPCR and (D) WB analysis of ERBB2 transcript and protein expression, respectively, in stage I/II vs. stage III/IV patient-derived cervical cancer tissues (n=43 stage I/II; n=22 Stage III/IV). Data were analyzed via Mann-Whitney U test. (E) RT-qPCR and (F) WB analysis of ERBB2 transcript and protein expression, respectively, in lymph node metastatic and non-metastatic patient-derived cervical cancer biopsies [n=46 lymph node (−); n=19 lymph node (+)]. Data were analyzed via Mann-Whitney U test. (G) Survival analysis using the Kaplan-Meier method according to high (above the median) or low (below the median) ERBB2 mRNA expression (n=32 in each cohort). The P-value was calculated using the log-rank test. For purposes of comparison across cohorts, the median ERBB2 mRNA and protein expression levels (normalized to the RT-qPCR housekeeping control and WB loading control GAPDH) in the normal cohort have been set to 1.0. Data in box plots are expressed as the median ± IQRs (boxes) and absolute ranges (whiskers). n=3. **P<0.01. RT-qPCR, reverse transcription-quantitative PCR; WB, western blotting; ERBB2, Erb-B2 Receptor Tyrosine Kinase 2; Pt, patient.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3′-UTR of human ERBB2 (ERBB2-3′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: Erb-B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53-responsive microRNA-3184-5p in cervical cancer cells

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: ERBB2 overexpression in cervical cancer cell lines stimulates viability, invasion and sphere-formation. (A) Confirmation of ERBB2 KD in siERBB2 cells and OE in ERBB2 vec cells via WB. GAPDH was used as the loading control. (B) Invasion of siERBB2 vs. siCtrl cells via Transwell assay. (C) Invasion of ERBB2 vec vs. Ctrl vec cells via Transwell assay. (D) Cellular viability of siERBB2, siCtrl, ERBB2 vec and Ctrl vec cells quantified using a Cell Counting Kit-8. (E) Sphere-formation of siERBB2 vs. siCtrl cells. (F) Sphere-formation of ERBB2 vec vs. Ctrl vec cells. (G) Analysis of metastasis-associated and cancer stem cell biomarkers mRNA and protein expression in siERBB2, siCtrl, ERBB2 vec and Ctrl vec cells via RT-qPCR and WB, respectively. GAPDH was used as the RT-qPCR housekeeping control and WB loading control. Data are expressed as the mean ± SEM (n=3). **P<0.01 vs. siCtrl or Ctrl vec analyzed via unpaired Student's t-test. KD, knockdown; OE, overexpression; WB, western blotting; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; Ctrl, control; vec, vector; ERBB2, Erb-B2 Receptor Tyrosine Kinase 2.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3′-UTR of human ERBB2 (ERBB2-3′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Over Expression, Transwell Assay, Cell Counting, Expressing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Oncology Reports

Article Title: Erb-B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53-responsive microRNA-3184-5p in cervical cancer cells

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: ERBB2 controls cervical cancer cell viability and invasion by regulating PIK3CA protein expression. (A) Schematic diagram of the ERBB2-ERRB3 complex interacting with PI3K(p85), thereby promoting the downstream phosphorylation of AKT and mTOR. (B) IP in cervical cancer cell lysates with antibodies against ERBB3 or IgG control. Expression levels of ERBB3, ERBB2 and PI3K(p85) in the IP fraction were assessed via WB. PIK3CA mRNA expression in transfected (C) HeLa and (D) SiHa cells assessed via RT-qPCR. GAPDH was used as the housekeeping control. PIK3CA, p-AKT/AKT and p-mTOR/mTOR protein expression in transfected (E) HeLa and (F) SiHa cells assessed via WB. GAPDH was used as the loading control. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit-8. (I) Sphere-formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. siCtrl or Ctrl vec; † P<0.05 and †† P<0.01 vs. siERBB2 or ERBB2 vec. Data were analyzed via one-way ANOVA. IP, immunoprecipitation; WB, western blotting; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; vec, vector; ERBB2, Erb-B2 Receptor Tyrosine Kinase 2; p-, phosphorylated; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3′-UTR of human ERBB2 (ERBB2-3′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Expressing, Transfection, Quantitative RT-PCR, Transwell Assay, Cell Counting, Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Oncology Reports

Article Title: Erb-B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53-responsive microRNA-3184-5p in cervical cancer cells

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: miR-3184-5p attenuates cervical cancer cell viability and invasion by targeting ERBB2. (A) Putative binding location for miR-3184-5p on ERBB2 3′-UTR via TargetScan analysis. (B) miR-3184-5p expression in HeLa and SiHa cervical cancer cell lines compared with in the non-cancerous human H8 cervical epithelial cell line assessed via RT-qPCR. U6 was used as the housekeeping control. **P<0.01 vs. H8; †† P<0.01 vs. SiHa. Luciferase reporter assay of ERBB2-3′-UTR WT or ERBB2-3′-UTR MU in (C) HeLa or (D) SiHa cells transfected with miR-3184-5p mimic or inhibitor, respectively. **P<0.01 vs. Ctrl mimic or Ctrl inhib. WB of (E) HeLa and (F) SiHa cells transfected with miR-3184-5p mimic or inhibitor, respectively. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit-8. (I) Sphere-formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). **P<0.01 vs. Ctrl vec; †† P<0.01 vs. miR-3184-5p mimic. Data were analyzed via one-way ANOVA. UTR, untranslated region; WT, wild-type; MU, mutant; Ctrl, control; inhib, inhibitor; WB, western blotting; RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA; vec, vector; ERBB2, Erb-B2 Receptor Tyrosine Kinase 2; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3′-UTR of human ERBB2 (ERBB2-3′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Transfection, Inhibition, Transwell Assay, Cell Counting, Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Oncology Reports

Article Title: Erb-B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53-responsive microRNA-3184-5p in cervical cancer cells

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: p53-activating Mithramycin A boosts miR-3184-5p expression, which lowers ERBB2 expression and attenuates viability and invasion of cervical cancer cell lines. (A) p53, p21 and ERBB2 protein expression in cervical cancer cultures incubated with MM or vehicle (DMSO) assessed via WB. GAPDH was used as the loading control. (B) miR-3184-5p expression in cervical cancer cultures incubated with MM or vehicle assessed via RT-qPCR. U6 was used as the housekeeping control. (C) p53 and ERBB2 protein expression in cervical cancer cultures transfected with a p53 overexpression plasmid or empty plasmid control assessed via WB. GAPDH was used as the loading control. (D) miR-3184-5p expression in cervical cancer cultures transfected with a p53 overexpression plasmid or empty plasmid control assessed via RT-qPCR. U6 was used as the housekeeping control. (E) Representative images of Transwell and sphere-formation assays in (E) HeLa and (F) SiHa cells, and quantitative analysis of viability, invasion and sphere-formation of cells treated with MM or vehicle. (G) Schematic diagram of the p53 activator MM rescuing miR-3184-5p expression, thereby suppressing ERBB2 transcription. This attenuates PIK3CA activity, which stimulates cervical cancer cell viability, invasion and sphere-formation. Data are expressed as the mean ± SEM (n=3). **P<0.01 analyzed via unpaired Student's t-test. MM, Mithramycin A; Ctrl, control; WB, western blotting; RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA; vec, vector; ERBB2, Erb-B2 Receptor Tyrosine Kinase 2; p-, phosphorylated; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3′-UTR of human ERBB2 (ERBB2-3′-UTR WT ; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChange™ Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3′-UTR WT to create the mutant (MU) ERBB2-3′-UTR MU .

Techniques: Expressing, Incubation, Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction