Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: Schematic diagram of ultrasound-mediated CRISPR/Cas9 gene editing of Cdh2 to inhibit tumor invasion and metastasis.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: CRISPR

Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: ( A ) Fluorescence images of Cas9- and EGFP-stably expressed 4T1 cells transfected with only pU6-sgRNA (EGFP)-mCherry (control), GPD, or GPD + US (GPD: the abbreviation of GVs-PEI-DNA). Scale bar = 200 μm; ( B ) determination of the transfection efficiency by flow cytometry through counting of the red-emitting cells; ( C ) quantitative analysis of the mCherry-expressed cells from ( B ) ( n = 3); ( D ) determination of the EGFP knockout efficiency ( n = 3). ns denotes p > 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: Fluorescence, Stable Transfection, Transfection, Flow Cytometry, Knock-Out

Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: ( A ) Fluorescence images of Cas9- and EGFP-stably expressed 4T1 cells transfected with only pU6-sgRNA (Cdh2)-mCherry (control), GPD, or GPD + US (GPD: the abbreviation of GVs–PEI–DNA). Scale bar = 100 μm; ( B ) determination of the transfection efficiency by flow cytometry through the counting of the red-emitting cells in EGFP-expressed cells; ( C ) quantitative analysis of the mCherry-expressed cells from ( B ) ( n = 3) *** p < 0.001. ( D ) Western blotting assay of the expression of N-cadherin in the wild-type (WT) and two Cdh2-KO cell lines. GAPDH was used as an internal marker.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: Fluorescence, Stable Transfection, Transfection, Flow Cytometry, Western Blot, Expressing, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Obesity and triple‐negative‐breast‐cancer: Is apelin a new key target?

doi: 10.1111/jcmm.15639

Figure Lengend Snippet: Infusion of obesity‐related levels of apelin favours TNBC brain metastatization. A, Ex vivo bioluminescent signals from 4T1 Luc‐GFP in lungs of PBS or apelin‐infused (0.1 µmol/kg/d) Balb/c nude mice. B, Quantification of bioluminescent signals of lung metastases by intensity. C, Ex vivo bioluminescent signals from 4T1 Luc‐GFP in brains of PBS or Apelin‐infused Balb/c nude mice. D, Quantification of metastatic brains in PBS or Apelin‐infused mice. Number of mice per group for (A, B): Control: 9, Apelin: 9, and for (C, D): Control: 9, Apelin: 10. Data were analysed using two‐way ANOVA followed by Bonferroni post hoc test for (B). Data were analysed using Fisher's exact test for (D)

Article Snippet: The 4T1 Luc‐GFP cell line was obtained from Perkin Elmer (4T1 Red FLuc‐GFP, PerkinElmer, Waltham, MA, USA) and grows only in immunodeficient mice.

Techniques: Ex Vivo

Journal: Advanced Biomedical Research

Article Title: Effect of Hydatid Cyst Fluid Antigens on Induction of Apoptosis on Breast Cancer Cells

doi: 10.4103/abr.abr_220_18

Figure Lengend Snippet: Effects of different antigens of hydatid cyst the 78 KD fraction (a), glycoprotein (b), crud hydatid cyst (c), glycolipid (d), antigen B (e), and control (f) on apoptosis and necrosis of 4T1 cells. The values of necrosis and apoptosis were shown in percentage (%)

Article Snippet: Breast cancer cell line (4T1) was obtained from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran).

Techniques: Control

Journal: Advanced Biomedical Research

Article Title: Effect of Hydatid Cyst Fluid Antigens on Induction of Apoptosis on Breast Cancer Cells

doi: 10.4103/abr.abr_220_18

Figure Lengend Snippet: Flow cytometry analysis of effects of different antigens of hydatid cyst the 78 KD fraction (a), glycoprotein (b), crud hydatid cyst (c), glycolipid (d), antigen B (e), and control (f) on apoptosis and necrosis of 4T1 cells

Article Snippet: Breast cancer cell line (4T1) was obtained from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran).

Techniques: Flow Cytometry, Control

Journal: Cancer research

Article Title: HDAC6 plays a non-canonical role in the regulation of anti-tumor immune responses, dissemination, and invasiveness of breast cancer.

doi: 10.1158/0008-5472.CAN-19-3738

Figure Lengend Snippet: (A) Growth kinetics of 4T1 implanted subcutaneously to the flank of female BALB/c mice, treated with different doses of NextA (1.0 to 25mg/kg), 5X/week. (B) Growth kinetics of 4T1 implanted orthotopically in 4th mammary gland, treated with different doses of NextA, 5X/week. (C) Quantification of lung nodules in vector control vs. 25mg/kg NextA treated mice, 5X/week. (D) Pictorial representation of lung nodules. (E) Tumor burden measured by Luciferin K-salt injected bioluminescent mice. (F) Quantification of surface nodules as counted per cubic mm of tumor volume. (G) Growth kinetics of non-target vs. HDAC6KD-4T1 tumors implanted orthotopically into mice. (H) Quantification of lung nodules from non-target vs. HDAC6KD-4T1 tumor-bearing host. (I) Pictorial representation of lung nodules.

Article Snippet: 4T1 cell line, a model for triple-negative murine carcinoma, was a generous gift from Dr. Scott Abrams’ laboratory at RPCI, Buffalo, NY (authenticated by IDEXX through STR-profiling: details provided as supplemental document ).

Techniques: Plasmid Preparation, Control, Injection

Journal: Cancer research

Article Title: HDAC6 plays a non-canonical role in the regulation of anti-tumor immune responses, dissemination, and invasiveness of breast cancer.

doi: 10.1158/0008-5472.CAN-19-3738

Figure Lengend Snippet: (A) Gene expression of EMT-molecules with or without in vitro NextA treatment (5μM) on 4T1 cells by qPCR. (B) Expression of EMT-genes with or without in vitro NextA (5μM) on metastatic 4T1 lung-isolated clones by qPCR. (C) Expression of EMT-genes after in vitro NextA treatment (5μM) on MDA-MB-231 cells by qPCR. (D) Expression of EMT-genes after in vitro NextA treatment (5μM) on MCF7 cells by qPCR. (E) Evaluation of E-Cad levels in 4T1 cells after in vitro NextA treatment (5μM) by western blot. Evaluation of acetylated tubulin as an internal indicator for NextA mediated acetylation function. (F) Detection of the endogenous levels of E-Cad by western blot from in vivo tumor samples treated with NextA (25mg/kg). (G) Endogenous expression of HDAC6 and E-Cad protein in non-target vs. HDAC6KD-4T1 clones. (H) Corresponding E-Cad RNA levels in non-target vs. HDAC6KD-4T1 clones.. (I) Expression of EMT-genes in non-target and HDAC6KD-4T1 cells. (J) NextA dose-dependent invasion by 4T1 cells measured in 2D invasion chamber. GM-6001, a broad-spectrum MMP-inhibitor, is used as a positive control. (K) Quantification of wound closure of 4T1 cells throughout 16h, with or without NextA, as analyzed by ImageJ. (L) Snapshots of wound closure of 4T1 cells throughout 16h, with or without NextA, captured on Leica DMi8 microscope with a piezo stage (Pecon) with phase-contrast.

Article Snippet: 4T1 cell line, a model for triple-negative murine carcinoma, was a generous gift from Dr. Scott Abrams’ laboratory at RPCI, Buffalo, NY (authenticated by IDEXX through STR-profiling: details provided as supplemental document ).

Techniques: Gene Expression, In Vitro, Expressing, Isolation, Clone Assay, Western Blot, In Vivo, Positive Control, Microscopy

Journal: Cancer research

Article Title: HDAC6 plays a non-canonical role in the regulation of anti-tumor immune responses, dissemination, and invasiveness of breast cancer.

doi: 10.1158/0008-5472.CAN-19-3738

Figure Lengend Snippet: (A) Growth kinetics of 4T1 tumors implanted in the 4th mammary gland of female BALB/c mice, treated with different doses of αPD-1 antibody (0.5 to 15mg/kg). (B) Quantification of lung nodules in vehicle control vs. αPD-1 treated mice. (C) Gene expression of IFNγ and PD-L1 in the in vivo tumors collected from the mice with or without αPD-1 treatment measured by qPCR. (D) Expression of PD-L1 gene in 4T1 cells treated in vitro with 5μM of NextA measured by qPCR. (E) Expression of PD-L1 on tumor cells co-cultured with CD3+ splenocytes from non-tumor bearing mice and αPD-1 antibody, with or without treatment with NextA or IFNγ-neutralizing antibody, measured by qPCR. (F) Expression of PD-L1 on tumor cells co-cultured with CD3+ splenocytes from 4T1 tumor-bearing mice and αPD-1 antibody, with or without treatment with NextA or IFNγ-neutralizing antibody, measured by qPCR.

Article Snippet: 4T1 cell line, a model for triple-negative murine carcinoma, was a generous gift from Dr. Scott Abrams’ laboratory at RPCI, Buffalo, NY (authenticated by IDEXX through STR-profiling: details provided as supplemental document ).

Techniques: Control, Gene Expression, In Vivo, Expressing, In Vitro, Cell Culture

Journal: Cancer research

Article Title: HDAC6 plays a non-canonical role in the regulation of anti-tumor immune responses, dissemination, and invasiveness of breast cancer.

doi: 10.1158/0008-5472.CAN-19-3738

Figure Lengend Snippet: (A) Schematic representation of combination treatment. (B) Growth kinetics of 4T1 tumors implanted in the 4th mammary gland of female BALB/c mice, treated with the predetermined doses of NextA and αPD-1; 6X/week. (C) Growth kinetics of the individual tumors in each treatment group. (D) Quantification of lung nodules from each treatment group. (E) Pictorial representation of lung nodules from each group.

Article Snippet: 4T1 cell line, a model for triple-negative murine carcinoma, was a generous gift from Dr. Scott Abrams’ laboratory at RPCI, Buffalo, NY (authenticated by IDEXX through STR-profiling: details provided as supplemental document ).

Techniques:

Journal: Cancer research

Article Title: HDAC6 plays a non-canonical role in the regulation of anti-tumor immune responses, dissemination, and invasiveness of breast cancer.

doi: 10.1158/0008-5472.CAN-19-3738

Figure Lengend Snippet: (A) Measurement of E-Cad gene expression by qPCR in 4T1 cells in vitro treated with E-Cad siRNA. (B) Measurement of E-Cad protein expression by Western blot in 4T1 cells in vitro treated with E-Cad siRNA. (C) Measurement of E-Cad protein expression by Western blot in 4T1 cells in vitro treated with E-Cad SiRNA and NextA (5μM). (D) Invasion capability of E-Cad silenced 4T1 cells at different concentrations of NextA (2.5 and 5μM) measured in 2D invasion chamber. (E) Expression of ZEB1 in the 4T1 cells after NextA (5μM) and Stattic (10μM) treatment, measured by qPCR. (F) MDA-MB-231 cells were treated with NextA (5μM), and the expression of E-Cad, ZEB1, and tubulin, evaluated by immunoblot. (G) A regulatory model is proposing HDAC6is as modulators of the STAT3/ZEB1 axis and subsequent regulation of the expression of E-Cadherin.

Article Snippet: 4T1 cell line, a model for triple-negative murine carcinoma, was a generous gift from Dr. Scott Abrams’ laboratory at RPCI, Buffalo, NY (authenticated by IDEXX through STR-profiling: details provided as supplemental document ).

Techniques: Gene Expression, In Vitro, Expressing, Western Blot