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: 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: Figure 1. 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'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Mann-Whitney U-Test, Comparison, Control, 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: Figure 2. 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'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Over Expression, Control, Transwell Assay, CCK-8 Assay, Expressing, Knockdown, Western Blot, 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: Figure 3. 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'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Phospho-proteomics, Control, Transfection, Transwell Assay, CCK-8 Assay, Immunoprecipitation, Western Blot, 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: Figure 4. 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'‑UTRWT or ERBB2‑3'‑UTRMU 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'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Binding Assay, Expressing, Control, Luciferase, Reporter Assay, Transfection, Inhibition, Transwell Assay, CCK-8 Assay, Mutagenesis, Western Blot, 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: Figure 5. 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'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Incubation, Control, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Western Blot

Journal: Cancers

Article Title: Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus

doi: 10.3390/cancers12113407

Figure Lengend Snippet: Molecular characterization of Sting knockout cancer cell lines. ( A ) Analysis of human HER2 display on cell surface of LLC1-HER2 (left) and CT26_HER2 (right) by FACS analysis; an unrelated antibody was used as negative control. ( B ) The graphic shows Tmem173 (transcript ID ENSMUST00000115728.4) gene organization. Full and empty boxes represent, respectively, coding and untranslated exons. The positions of guide RNAs used for CRISPR/Cas9 genome editing to generate Sting knockout cancer cell lines are indicated by arrows. ( C ) Western blot analysis of Sting protein in CT26-HER2, LLC1_HER2 and their Sting knockout cell lines counterparts. Gamma tubulin was used as standard. ( D ) PCR screening of CT26-HER2_SKO and LLC1_HER2_SKO cell lines to assess the absence of eGFP and Cas9 residues in genomic DNA. Cas9/eGFP-encoding vector was used as positive control (C+). Genomic DNA from parental CT26-HER2 and LLC-HER2 cell lines was used as negative control (C−). ( E ) Cell doubling per day were assessed for Sting wild-type (grey lines) and Sting knockout (black lines) LLC1 (left) and CT26 (right) cell lines. The differences in cell doubling were calculated by Student’s t -test and were not statistically significant (Ns) to each passage.

Article Snippet: Human HER2 transduction of CT26 cells was performed by Origene, Rockville, MD, USA, RC212583L1V.

Techniques: Knock-Out, Negative Control, CRISPR, Western Blot, Plasmid Preparation, Positive Control

Journal: Cancers

Article Title: Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus

doi: 10.3390/cancers12113407

Figure Lengend Snippet: Comparison of viral effectiveness in Sting knockout vs. parental wild-type cancer cell lines. ( A , B ) Spread of eGFP-encoding R-LM113 was evaluated by fluorescence microscopy in STING wild-type and knockout LLC1 (5×) ( A ) and CT26 (10×) ( B ) cell lines. ( C ) The lytic activity of R-LM113 was evaluated by extracellular LDH (lactate dehydrogenase) release in cell supernatants over the time course of infection (72, 96 and 120 h) in LLC1-HER2 (grey lines) and LLC1-HER2_SKO (black lines) at two different concentrations of viral particles (1 multiplicity of infection (MOI) continuous lines and 0.5 MOI dashed lines). ( D ) The same experiments performed in panel C were recapitulated in CT26-HER2 and CT26-HER2_SKO. All the infections were performed as biological replicates. The statistical significances for experiments described in panel c and d were calculated by Student’s t -test comparing MOI-matched Sting wild-type vs. knockout cell lines. The p -values were 0.00115 and 0.000219, respectively, for 1 and 0.5 MOI in panel C; 0.01583, 0.008543, respectively, for 1 and 0.5 MOI in panel D. ( E , F ) Evaluation of viral replication of R-LM113 in Sting wild-type and knockout LLC1 ( E ) and CT26 ( F ) infected with 0.3 PFU/cell. The qPCR-TaqMan analysis revealed the genome copies per mL (gc/mL) produced by the virus over time (24, 48, 72 h for LLC1 and 72, 96, 20 h for CT26). The statistical significances for experiments described in panel e and f were calculated by Student’s t -test comparing Sting wild-type vs. knockout cell lines. The p -values calculated on biological replicates were 0.0013 for LLC1 cell line and 0.0005 for CT26 cell line. ( G , H ) Analysis of the R-LM113 viral titers obtained in Sting wild-type and knockout LLC1 ( G ) and CT26 ( H ) cells infected with 0.3 PFU/cell. Plaque assay was performed as biological replicate. The statistical significance for experiments described in panel g and h was calculated by Student’s t -test comparing Sting wild-type vs. knockout cell lines. The p -values were 0.038 for LLC1 cell line and 0.02 for CT26 cell line. p < 0.05 *; p < 0.005 **; p < 0.0005 ***.

Article Snippet: Human HER2 transduction of CT26 cells was performed by Origene, Rockville, MD, USA, RC212583L1V.

Techniques: Knock-Out, Fluorescence, Microscopy, Activity Assay, Infection, Produced, Plaque Assay

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: HER2 overexpression is a driver for invasive progression of DCIS (A) Representative IHC images of p-ERK and p-AKT. (B) Plotted H scores for p-ERK and p-AKT expression in HER2 + DCIS and HER2 – DCIS controls. (C) Tumor-free survival curves of responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (D) Tumor-free survival curves of non-responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (E) Representative whole-mount images and Ku80-stained sections of DCIS-injected mammary glands from DCIS063 mice treated with vehicle (left panels) or herceptin (right panels). (F) Schematic representation of the experimental setup for lentiviral overexpression of HER2 in HER2 – DCIS cells. (G) Western blot showing expression of HER2 and phospho-HER2 in parental 293T cells and 293T cells transduced with the HER2-GFP lentivirus. (H) Representative whole-mount images of intraductally injected mammary glands, showing non-invasive replacement growth of non-transduced HER2 – DCIS cells (left panel) and invasive expansive growth of the same DCIS cells transduced with the HER2-GFP lentivirus (right panel). Inset shows lesion without αSMA marker indicating GFP expression (green). Cells of human origin are marked with Ku80 (gray), myoepithelial cells are marked with αSMA (magenta). Inset shows lesion without αSMA marker indicating HER2-GFP expression (green). See also Figure S7 .

Article Snippet: Primary or PDX DCIS tissue was digested to a single-cell solution as described before and was transfected with a lentivirus (ERBB2-GFP lentivirus, Origene, RC222909L4, or Akaluciferase, courtesy of the lab of Shinae Kizaka-Kondo cloned into the pRRL vector backbone, Addgene plasmid #31485) or vehicle by putting the cells and the virus in a falcon tube and spin for 2 hours at 2500 rpm at RT.

Techniques: Over Expression, Expressing, Staining, Injection, Western Blot, Transduction, Marker

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: A living biobank of MIND-PDX models of DCIS retaining histological and molecular features of primary lesions (A) Schematic overview of the generation and characterization of 130 MIND-PDX models of DCIS. (B) Pie chart of the grade distribution of the primary DCIS lesions. (C) Pie chart of the DCIS take rate in MIND-PDX models. (D) Examples of H&E-stained sections of the different growth patterns observed in primary DCIS lesions (top row) or DCIS-MIND outgrowths (middle row). Bottom row: human-specific Ku80 staining showing that DCIS-MIND outgrowths have a human origin. (E) Distribution of growth patterns of primary DCIS lesions and the corresponding DCIS-MIND outgrowths. A black line indicates concordance between primary and PDX, whereas an orange line indicates a discordance between primary and PDX. (F) Growth pattern analyses between an early (3–6 months) and late (12 months) time points. (G) Examples of immunohistochemistry for ER, PR, HER2, and Ki67 expression in DCIS-MIND lesions (top row) vs. matched primary DCIS lesions (bottom row). (H) Distribution of molecular subtypes (luminal A: ER + , PR +/− , HER2 − , Ki67 < 20%; luminal B: ER + , PR +/− , HER2 – , Ki67 ≥ 20%; HER2 + : ER +/− , PR +/− , and HER2 + ; basal: ER − , PR − , HER2 − ) of primary DCIS lesions and the corresponding DCIS-MIND lesions. A black line indicates concordance between primary and PDX, whereas an orange line indicates a discordance between primary and PDX. See also Figure S1 and Table S1 .

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

Techniques: Staining, Immunohistochemistry, Expressing

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: DCIS-MIND models retain mutational and transcriptional features of the primary DCIS lesions (A) Oncoprint showing the mutational landscape of the primary DCIS lesions, including amplifications, single-nucleotide variants, and insertion-deletions (indels) for the top mutated genes in our breast cancer gene panel. Annotations for each model includes ER, PR, and HER2 status. (B) Oncoprint showing amplifications, single-nucleotide variants, and insertion-deletions (indels) in cancer genes in primary DCIS lesions and corresponding DCIS-MIND lesions for the top mutated genes. (C) Unsupervised clustering of DCIS-MIND lesions based on PAM50 genes, showing clustering of luminal, HER2 + , and basal-like DCIS lesions. Annotations include origin (primary or PDX) and molecular subtype based on PAM50 or IHC. (D) Unsupervised clustering of DCIS-MIND lesions based on 90 informative genes resulting in three DCIS subtypes proposed by Strand et al. See also Figure S2 and Tables S2 and .

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

Techniques:

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: Comparison of clinical biomarkers related to invasive progression of DCIS (A) Whole-mount analysis (top row), H&E staining (middle row), and human-specific Ku80 staining (bottom row) of DCIS-MIND lesions showing non-invasive growth (left panel) or invasive growth (right panel). Cells of human origin are marked with Ku80 (green), myoepithelial cells are marked with alpha-smooth muscle actin (αSMA) (magenta). Blue arrows/circles indicate invasive cells. (B) Pie chart of the percentage of DCIS-MIND lesions with non-invasive growth, micro-invasion, or invasive progression. (C) Odds ratio table showing risk scores (univariate and multivariate linear regression models) for association between common clinical parameters, DCIS subtypes as proposed by Strand et al., and multiple invasive recurrence classifiers for invasive progression of DCIS-MIND lesions, identifying HER2 overexpression and solid growth patterns as independent risk factors. Molecular subtype and growth pattern parameters are based on DCIS-MIND characteristics while the other parameters are based on patient characteristics. See also Figure S3 .

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

Techniques: Comparison, Clinical Proteomics, Staining, Over Expression

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: HER2 overexpression is a driver for invasive progression of DCIS (A) Representative IHC images of p-ERK and p-AKT. (B) Plotted H scores for p-ERK and p-AKT expression in HER2 + DCIS and HER2 – DCIS controls. (C) Tumor-free survival curves of responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (D) Tumor-free survival curves of non-responsive HER2 + DCIS models treated with vehicle (C) or herceptin (T) with log rank test. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. (E) Representative whole-mount images and Ku80-stained sections of DCIS-injected mammary glands from DCIS063 mice treated with vehicle (left panels) or herceptin (right panels). (F) Schematic representation of the experimental setup for lentiviral overexpression of HER2 in HER2 – DCIS cells. (G) Western blot showing expression of HER2 and phospho-HER2 in parental 293T cells and 293T cells transduced with the HER2-GFP lentivirus. (H) Representative whole-mount images of intraductally injected mammary glands, showing non-invasive replacement growth of non-transduced HER2 – DCIS cells (left panel) and invasive expansive growth of the same DCIS cells transduced with the HER2-GFP lentivirus (right panel). Inset shows lesion without αSMA marker indicating GFP expression (green). Cells of human origin are marked with Ku80 (gray), myoepithelial cells are marked with αSMA (magenta). Inset shows lesion without αSMA marker indicating HER2-GFP expression (green). See also Figure S7 .

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

Techniques: Over Expression, Expressing, Staining, Injection, Western Blot, Transduction, Marker

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, Polymer, 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: Figure 1. 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: SiHa cells were transfected with plasmid vectors, while HeLa cells were transfected with small-interfering RNAs (siRNAs). pCMV6-XL4/5 plasmid vectors containing the TrueClone® human cDNA sequences for human TP53 (NM_000546; cat. no. SC119832), PIK3CA (NM_006218; cat. no. SC116227) or ERBB2 (NM_004448; cat. no. SC128161), and an empty negative control (NC) plasmid vector (cat. no. PCMV6XL5) were obtained from OriGene Technologies, Inc. For plasmid transfections, cells were seeded in 6-well plates (1.5x105 cells/well) and transiently transfected with 1 μg plasmid using Lipofectamine® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min at room temperature and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. siRNAs targeting human ERBB2 (ERBB2-siRNA; cat. no. sc-29405), human PIK3CA (PIK3CA-siRNA; cat. no. sc-39127) and scrambled control (scr-siRNA; cat. no. sc-37007) were obtained from Santa Cruz Biotechnology, Inc. For siRNA transfections, cells were seeded in 6-well plates (2x105 cells/well) and transiently transfected with 80 pmol siRNA using Lipofectamine 3000 transfection reagent for 7 h at 37 ̊C and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. miR-3184-5p (miRBase accession no. MIMAT0015064) mirVana® miRNA mimic (cat. no. 4464066) and mirVana® miRNA inhibitor (cat. no. 4464084), as well as the corresponding controls (cat. nos.

Techniques: Expressing, Mann-Whitney U-Test, Comparison, Control, 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: Figure 2. 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: SiHa cells were transfected with plasmid vectors, while HeLa cells were transfected with small-interfering RNAs (siRNAs). pCMV6-XL4/5 plasmid vectors containing the TrueClone® human cDNA sequences for human TP53 (NM_000546; cat. no. SC119832), PIK3CA (NM_006218; cat. no. SC116227) or ERBB2 (NM_004448; cat. no. SC128161), and an empty negative control (NC) plasmid vector (cat. no. PCMV6XL5) were obtained from OriGene Technologies, Inc. For plasmid transfections, cells were seeded in 6-well plates (1.5x105 cells/well) and transiently transfected with 1 μg plasmid using Lipofectamine® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min at room temperature and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. siRNAs targeting human ERBB2 (ERBB2-siRNA; cat. no. sc-29405), human PIK3CA (PIK3CA-siRNA; cat. no. sc-39127) and scrambled control (scr-siRNA; cat. no. sc-37007) were obtained from Santa Cruz Biotechnology, Inc. For siRNA transfections, cells were seeded in 6-well plates (2x105 cells/well) and transiently transfected with 80 pmol siRNA using Lipofectamine 3000 transfection reagent for 7 h at 37 ̊C and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. miR-3184-5p (miRBase accession no. MIMAT0015064) mirVana® miRNA mimic (cat. no. 4464066) and mirVana® miRNA inhibitor (cat. no. 4464084), as well as the corresponding controls (cat. nos.

Techniques: Over Expression, Control, Transwell Assay, CCK-8 Assay, Expressing, Knockdown, Western Blot, 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: Figure 3. 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: SiHa cells were transfected with plasmid vectors, while HeLa cells were transfected with small-interfering RNAs (siRNAs). pCMV6-XL4/5 plasmid vectors containing the TrueClone® human cDNA sequences for human TP53 (NM_000546; cat. no. SC119832), PIK3CA (NM_006218; cat. no. SC116227) or ERBB2 (NM_004448; cat. no. SC128161), and an empty negative control (NC) plasmid vector (cat. no. PCMV6XL5) were obtained from OriGene Technologies, Inc. For plasmid transfections, cells were seeded in 6-well plates (1.5x105 cells/well) and transiently transfected with 1 μg plasmid using Lipofectamine® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min at room temperature and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. siRNAs targeting human ERBB2 (ERBB2-siRNA; cat. no. sc-29405), human PIK3CA (PIK3CA-siRNA; cat. no. sc-39127) and scrambled control (scr-siRNA; cat. no. sc-37007) were obtained from Santa Cruz Biotechnology, Inc. For siRNA transfections, cells were seeded in 6-well plates (2x105 cells/well) and transiently transfected with 80 pmol siRNA using Lipofectamine 3000 transfection reagent for 7 h at 37 ̊C and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. miR-3184-5p (miRBase accession no. MIMAT0015064) mirVana® miRNA mimic (cat. no. 4464066) and mirVana® miRNA inhibitor (cat. no. 4464084), as well as the corresponding controls (cat. nos.

Techniques: Expressing, Phospho-proteomics, Control, Transfection, Transwell Assay, CCK-8 Assay, Immunoprecipitation, Western Blot, 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: Figure 4. 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'‑UTRWT or ERBB2‑3'‑UTRMU 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: SiHa cells were transfected with plasmid vectors, while HeLa cells were transfected with small-interfering RNAs (siRNAs). pCMV6-XL4/5 plasmid vectors containing the TrueClone® human cDNA sequences for human TP53 (NM_000546; cat. no. SC119832), PIK3CA (NM_006218; cat. no. SC116227) or ERBB2 (NM_004448; cat. no. SC128161), and an empty negative control (NC) plasmid vector (cat. no. PCMV6XL5) were obtained from OriGene Technologies, Inc. For plasmid transfections, cells were seeded in 6-well plates (1.5x105 cells/well) and transiently transfected with 1 μg plasmid using Lipofectamine® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min at room temperature and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. siRNAs targeting human ERBB2 (ERBB2-siRNA; cat. no. sc-29405), human PIK3CA (PIK3CA-siRNA; cat. no. sc-39127) and scrambled control (scr-siRNA; cat. no. sc-37007) were obtained from Santa Cruz Biotechnology, Inc. For siRNA transfections, cells were seeded in 6-well plates (2x105 cells/well) and transiently transfected with 80 pmol siRNA using Lipofectamine 3000 transfection reagent for 7 h at 37 ̊C and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. miR-3184-5p (miRBase accession no. MIMAT0015064) mirVana® miRNA mimic (cat. no. 4464066) and mirVana® miRNA inhibitor (cat. no. 4464084), as well as the corresponding controls (cat. nos.

Techniques: Binding Assay, Expressing, Control, Luciferase, Reporter Assay, Transfection, Inhibition, Transwell Assay, CCK-8 Assay, Mutagenesis, Western Blot, 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: Figure 5. 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: SiHa cells were transfected with plasmid vectors, while HeLa cells were transfected with small-interfering RNAs (siRNAs). pCMV6-XL4/5 plasmid vectors containing the TrueClone® human cDNA sequences for human TP53 (NM_000546; cat. no. SC119832), PIK3CA (NM_006218; cat. no. SC116227) or ERBB2 (NM_004448; cat. no. SC128161), and an empty negative control (NC) plasmid vector (cat. no. PCMV6XL5) were obtained from OriGene Technologies, Inc. For plasmid transfections, cells were seeded in 6-well plates (1.5x105 cells/well) and transiently transfected with 1 μg plasmid using Lipofectamine® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min at room temperature and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. siRNAs targeting human ERBB2 (ERBB2-siRNA; cat. no. sc-29405), human PIK3CA (PIK3CA-siRNA; cat. no. sc-39127) and scrambled control (scr-siRNA; cat. no. sc-37007) were obtained from Santa Cruz Biotechnology, Inc. For siRNA transfections, cells were seeded in 6-well plates (2x105 cells/well) and transiently transfected with 80 pmol siRNA using Lipofectamine 3000 transfection reagent for 7 h at 37 ̊C and then incubated in fresh medium at 37 ̊C for an additional 48 h prior to subsequent experimentation. miR-3184-5p (miRBase accession no. MIMAT0015064) mirVana® miRNA mimic (cat. no. 4464066) and mirVana® miRNA inhibitor (cat. no. 4464084), as well as the corresponding controls (cat. nos.

Techniques: Expressing, Incubation, Control, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Western Blot

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