Journal: eNeuro

Article Title: The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent

doi: 10.1523/ENEURO.0272-17.2017

Figure Lengend Snippet: List of antibodies used for RPPA analysis

Article Snippet: ErbB2/HER2(Y1248)_R_V , Imgenex , IMG-90189 , Rabbit.

Techniques: Plasmid Preparation

Journal: BioMed Research International

Article Title: Tongxinluo Attenuates Myocardiac Fibrosis after Acute Myocardial Infarction in Rats via Inhibition of Endothelial-to-Mesenchymal Transition

doi: 10.1155/2019/6595437

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Anti-phospho-ErbB2 , Abcam, ab47262 , 1:500.

Techniques:

Journal: BioMed Research International

Article Title: Tongxinluo Attenuates Myocardiac Fibrosis after Acute Myocardial Infarction in Rats via Inhibition of Endothelial-to-Mesenchymal Transition

doi: 10.1155/2019/6595437

Figure Lengend Snippet: NRG-1/ErbB-PI3K/AKT signaling pathway was activated by TXL . Effects of TXL on protein expression of NRG-1, p-ErbB2, p-ErbB4, p-AKT, and snail in vitro were determined by Western blotting analysis. (a1)–(a6) showed corresponding column chart. β -actin expressions served as internal control. n=3, ∗∗ P<0.01 vs. control group; # P<0.05 ## P<0.01 vs. hypoxia group; & P<0.05 && P<0.01 vs. hypoxia +NRG-1 siRNA group; + P<0.05 ++ P<0.01 vs. hypoxia +TXL group.

Article Snippet: Anti-phospho-ErbB2 , Abcam, ab47262 , 1:500.

Techniques: Expressing, In Vitro, Western Blot

Journal: ACS Omega

Article Title: Nanolipoprotein-Mediated Her2 Protein Transfection Induces Malignant Transformation in Human Breast Acinar Cultures

doi: 10.1021/acsomega.1c03086

Figure Lengend Snippet: Her2 carried by NLPs causes malignant-like organization in nonmalignant cells. (A) Nonmalignant cells cultured in lrECM form growth arrested polarized acinar-like structure with the basal organization of α6 integrin (green) and lateral organization of β-catenin (red). (B) Malignant breast cancer cells form disorganized structures with no cell polarity, which fail to growth arrest, as shown by the presence of mitotic figures (white arrow). (C) Nonmalignant cells treated with Her2-NLPs form apolar masses, which fail to growth arrest. (D) In contrast, nonmalignant cells treated with empty NLPs organize normally. (E) Nonmalignant cells treated with Her2-NLPs and a Her2 dimerization inhibitory antibody organize normally. (F) Significantly more structures organize well in nonmalignant, empty-NLP-treated, inhibitory antibody only, and Her2-NLP+ inhibitory antibody conditions, whereas Her2-NLP-treated and malignant cells are less likely to organize into polarized, growth-arrested acini ( n = 5 biological replicates, * indicates post hoc test significance of p < 0.05, ** indicates post hoc test of p < 0.001). (G) Her2-NLPs show dose–response behavior, with fewer structures organizing well with increasing NLP dosage ( n = 3 biological replicates, * indicates post hoc test significance of p < 0.05, ** indicates post hoc test of p < 0.001). (H) Her2-NLP-treated structures are more likely to contain a mitotic figure than untreated or empty-NLP-treated cells ( n = 3 biological replicates, * indicates post hoc test significance of p < 0.05, ** indicates post hoc test of p < 0.001). (I) Increasing the doses of Her2-NLPs from 0.5 to 25 μg/mL causes increasing abnormalities in the acinar structure.

Article Snippet: Cells in lrECM were transferred to dishes, allowed to gel for 20 min at 37 °C, then covered with complete culture media ± 5 μg/mL Her2-NLPs or empty NLPs or Her2-NLPs plus a Her2 blocking antibody (clone 4D5, MCA6092, Biorad) at 26.67 μg/mL (which represented 1:1 molar ratio with Her2-NLPs).

Techniques: Cell Culture

Journal: ACS Omega

Article Title: Nanolipoprotein-Mediated Her2 Protein Transfection Induces Malignant Transformation in Human Breast Acinar Cultures

doi: 10.1021/acsomega.1c03086

Figure Lengend Snippet: Her2-NLPs induce gene expression changes in nonmalignant 3D cultures. (A) PCA plot for all sample sequences shows that principal component 1 separates biological replicates, and principal component 2 separates Her2-NLP treated from other conditions. (B) Genes common to multiple comparisons include BCRYN1 for all NLP-treated cells versus untreated samples; and CCL5, CUX2, EBI3, and TGM2 for Her2-NLP-treated samples versus either empty-NLP or untreated samples. (C) Ingenuity pathway analysis (IPA) network comparing Her2-NLPs to empty-NLP disks predicts relationships among several biological families and transcriptional regulators involved in cancer progression and metastasis, including (D) ERK, Akt, p38MAPK, and STAT3. Genes in red indicate hits found in the dataset. Orange coloring indicates predicted activation of biomolecules, and blue indicates predicted inhibition. Deeper color saturation indicates more confidence in predicted regulation. (E) Secondary tumor formation is the top activated disease network associated with Her2-NLP treatment compared to untreated samples. (F) Organ degeneration is predicted to be inhibited with Her2-NLP treatment compared to untreated samples. Genes in red indicate upregulated genes found in the dataset, whereas genes in purple indicate those that were downregulated in the dataset. Orange coloring indicates predicted activation, and blue indicates predicted inhibition of the biological phenotype. Log 2 fold change ±, p < 0.05.

Article Snippet: Cells in lrECM were transferred to dishes, allowed to gel for 20 min at 37 °C, then covered with complete culture media ± 5 μg/mL Her2-NLPs or empty NLPs or Her2-NLPs plus a Her2 blocking antibody (clone 4D5, MCA6092, Biorad) at 26.67 μg/mL (which represented 1:1 molar ratio with Her2-NLPs).

Techniques: Expressing, Activation Assay, Inhibition

Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Plasma levels of Foxp3. Peripheral blood mononuclear cells were collected from each group, and the proportions of CD4 + CD25 + Foxp3 + T reg /CD4 + T-cells were analyzed by flow cytometry, and quantified using FlowJo 7.6.1 software. (A) Histogram. Data are presented as the mean ± standard deviation. *P<0.01, vs. the AS group. (B) Negative group, (C) AS group, (D) atorvastatin group and (E) IL-35 group. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35; APC, allophycocyanin; PE, phycoerythrin.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Clinical Proteomics, Flow Cytometry, Software, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Levels of Foxp3 in atherosclerotic lesions. The deposition of Foxp3 in arteries from various groups was detected by immunohistochemistry (magnification, ×400). Positive Foxp3 was shown as brown nuclei. In the (A) negative and (B) AS groups, the deposition of Foxp3 in the lesions was minimal. Conversely, Foxp3 deposition was increased in the lesions of the (C) atorvastatin and (D) IL-35 groups, as compared with the AS group. (E) This was shown to be significant following quantification. There was no significant difference in the levels of Foxp3 between the atorvastatin and IL-35 groups. *P<0.01, vs. the negative control. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Immunohistochemistry, Negative Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Peptidomimetic-based antibody surrogate for HER2

doi: 10.1016/j.apsb.2021.04.016

Figure Lengend Snippet: (A) Schematic presentation of one-bead-two-compounds TentalGel beads for library. (B) Scheme for the synthesis of OBTC cyclic γ -AApeptides library. (a) Soak in water for overnight; (b) (Boc) 2 O, DCM/ether; (c) Fmoc-Met-OH, HOBt, DIC, DMF; (d) deprotect Fmoc by 20% piperidine in DMF; (e) split into five portions equally; (f) Dde protected amino acids, PyBOP, NEM, DMF; (g) deprotect Boc by TFA/triisopropylsilane/H 2 O/Thioanisole (94:2:2:2); (h) Fmoc protected γ -AApeptides, HOBt, DIC, DMF; (i) deprotecting alloc by Pd(PPh 3 ) 4 and Me 2 NH·BH 3 in DCM; (j) Dmt protected mercaptopropionic acid, HOBt, DIC, DMF; (k) deprotecting Dde by NH 2 OH . HCl and imidazole in NMP/DCM (5:1); (l) split-and-pool synthesis, repeating the previous steps; (m) deprotecting Fmoc by 20% piperidine in DMF; (n) 4-(bromomethyl)benzoyl chloride, DIPEA, DCM; (o) deprotecting Dmt by TFA/triisopropylsilane/DCM (2:2:96); (p) (NH 4 ) 2 CO 3 , DMF/H 2 O (1:1). Xs are regular α -amino acids. (C) Scheme showing the overall strategy involved in library screening against HER2.

Article Snippet: HER2 was purchased from Creative BioMart; Anti-phospho-HER2 antibody was purchased from Thermo Fisher Scientific; Anti-phospho-AKT and Anti-phospho-ERK antibodies were from Cell Signaling Technology; GAPDH loading control monoclonal antibody was purchased from Invitrogen.

Techniques: Library Screening

Journal: Acta Pharmaceutica Sinica. B

Article Title: Peptidomimetic-based antibody surrogate for HER2

doi: 10.1016/j.apsb.2021.04.016

Figure Lengend Snippet: (A) Signal transduction pathway mediated by HER2 and proposed inhibition by cyclic peptides monomer and dimer. (B) Western blot analyses of SKBR3 cell lysates following M-3-6 incubation in vitro . M-3-6 treatment resulted in a dose-dependent inhibition of HER2 receptor phosphorylation and a reduction in the phosphorylation of AKT and ERK, downstream signaling of HER2. (C) Western blot analyses of SKBR3 cell lysates following M-3-6-D incubation in vitro . M-3-6-D treatment resulted in a much more effective phosphorylation inhibition compared with M-3-6. (D) M-3-6 and M-3-6-D inhibit cell proliferation. EGF-driven proliferation of SKBR3 cells was analyzed by CCK-8 proliferation assay. EGF (100 ng/mL) induced the proliferation of cells under serum-starved conditions ( ### P < 0.001 vs . non-stimulated cells), and both M-3-6 and M-3-6-D inhibited EGF-induced proliferation significantly in a dose-dependent manner (∗ P < 0.05 vs . EGF-stimulated cells, ∗∗∗ P < 0.001 vs . EGF-stimulated cells).

Article Snippet: HER2 was purchased from Creative BioMart; Anti-phospho-HER2 antibody was purchased from Thermo Fisher Scientific; Anti-phospho-AKT and Anti-phospho-ERK antibodies were from Cell Signaling Technology; GAPDH loading control monoclonal antibody was purchased from Invitrogen.

Techniques: Transduction, Inhibition, Western Blot, Incubation, In Vitro, Phospho-proteomics, CCK-8 Assay, Proliferation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Peptidomimetic-based antibody surrogate for HER2

doi: 10.1016/j.apsb.2021.04.016

Figure Lengend Snippet: Therapeutic efficacy of M-3-6 and M-3-6-D in SKBR3 xenograft models. (A) Mice body weight shift curve of the mice during the experiment. Arrows indicate the time of compounds treatment. (B) Time course assessment of total tumor volume. The day when treatment started was recorded as day 0 and arrows indicate the time of injection. After 2 weeks injection, tumor volume was measured once every 3 days until Day 24. (C) Immunohistochemical staining for P-HER2, P-AKT and P-ERK. Representative staining of section from SKBR3 tumors with antibodies against the indicated proteins. Scale bar: 100 nm.

Article Snippet: HER2 was purchased from Creative BioMart; Anti-phospho-HER2 antibody was purchased from Thermo Fisher Scientific; Anti-phospho-AKT and Anti-phospho-ERK antibodies were from Cell Signaling Technology; GAPDH loading control monoclonal antibody was purchased from Invitrogen.

Techniques: Drug discovery, Injection, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

doi: 10.18632/oncotarget.7273

Figure Lengend Snippet: The percentages of live and apoptotic cells were determined by FACS analysis (see Materials and Methods section). ( A ) Representative dot plot of FACS analysis of treated PC14 cells stained with annexin-V FITS and PI. PC14 singlet-cell events are distinguished from target cells by the CD340 specific marker. Numbers in the quadrants are the percentages of PC14 cells within each quadrant. ( B , C ) Quantitative results of FACS analysis. B. Percentage of live (low PI, low annexin V) PC14 cells cultured alone, with astrocytes, alone plus Taxol, with astrocytes in a contact-dependent manner (mixed co-culture) plus Taxol, and with astrocytes in a contact-independent manner (separated by a transwell membrane (TW)) plus Taxol. Results are expressed as percentages of untreated PC14 cells. C. Apoptotic (high annexin V, low PI) PC14 cells cultured alone, with astrocytes, alone plus Taxol, with astrocytes in a contact-dependent manner plus Taxol, and with astrocytes in a transwell (TW) plus Taxol. The results are expressed as percentage of total cells and are presented as means ± SEM. Analysis (One-way ANOVA) revealed significant differences in live cell percentages and apoptotic cells between annexin V-stained and PI-stained cells, P < 0.05. Post-hoc analysis was performed by Fisher's LSD: * P < 0.05, ** P = 0.01, **** P < 0.0001; n = 4.

Article Snippet: The cells were incubated at 37°C in a humidified 5% CO 2 incubator for 6 h, harvested, and stained for 15 min at RT with APC anti-human CD340 Ab to label PC14 cells and with anti-mouse GLAST-PE conjugated antibody (Miltenyi Biotec) to label astrocytes.

Techniques: Staining, Marker, Cell Culture, Co-Culture Assay, Membrane

Journal: Oncotarget

Article Title: Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

doi: 10.18632/oncotarget.7273

Figure Lengend Snippet: PC14 cells (CD340 positive) containing 22bpCy3 were assayed by FACS analysis (see Materials and Methods section). ( A ) Time course of 22bpCy3 transfer from astrocytes to PC14 cells. B−G. Representative dot plots of the results of FACS analysis after the different treatments, depicting total live-cell gate records. ( B ) PC14 cells (CD340 positive) alone. ( C ) 22bpCy3-transfected astrocytes (Cy3 positive) alone; positive for Cy3. D, E. PC14 cells co-cultured with astrocytes-22bpCy3 in a contact-dependent ( D ) or contact-independent (transwell, TW) manner ( E ) F, G. Astrocytes-22bpCy3 were co-cultured with PC14 cells that were untreated (control) ( F ) or treated with RNase A/T ( G ) ( H ) Quantitative results of FACS analysis, expressed as mean fluorescence intensity (MFI) of 22bpCy3 recorded in the PC14 cells and presented as means ± SEM. Analysis (One-way ANOVA) revealed significant differences in MFI of 22bpCy3 between the groups: P < 0.05. Post-hoc analysis by Fisher's LSD yielded * P < 0.05, ** P < 0.01, **** P < 0.0001; n = 5.

Article Snippet: The cells were incubated at 37°C in a humidified 5% CO 2 incubator for 6 h, harvested, and stained for 15 min at RT with APC anti-human CD340 Ab to label PC14 cells and with anti-mouse GLAST-PE conjugated antibody (Miltenyi Biotec) to label astrocytes.

Techniques: Transfection, Cell Culture, Control, Fluorescence

Journal: Oncotarget

Article Title: Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

doi: 10.18632/oncotarget.7273

Figure Lengend Snippet: Astrocytes-22bpCy3 were co-cultured with MDA-MB-231 cells for 6 h. MDA-MB-231 cells (CD340 positive) containing 22bpCy3 were assayed by FACS analysis (see Materials and Methods section). ( A − E ) Representative dot plots of the results of FACS analysis after the different treatments, depicting total live-cell gate records A. MDA-MB-231 cells (positive for CD340) alone. B. 22bpCy3-transfected astrocytes (Cy3-positive) alone. C, D. MDA-MB-231 cells co-cultured with astrocytes-22bpCy3 in a contact-dependent (C) or contact-independent (transwell, TW) manner (D). E. MDA-MB-231 cells co-cultured with astrocytes-22bpCy3 treated with RNase A/T. ( F ) Quantitative results of the FACS analysis, expressed as MFI of 22bpCy3 recorded in the MDA-MB-231 cells and presented as means ± SEM. Analysis (One-way ANOVA) revealed significant differences in MFI of 22bpCy3 between the groups: P < 0.01. Post-hoc analysis by Fisher's LSD yielded ** P < 0.01; n = 5.

Article Snippet: The cells were incubated at 37°C in a humidified 5% CO 2 incubator for 6 h, harvested, and stained for 15 min at RT with APC anti-human CD340 Ab to label PC14 cells and with anti-mouse GLAST-PE conjugated antibody (Miltenyi Biotec) to label astrocytes.

Techniques: Cell Culture, Transfection

Journal: Oncotarget

Article Title: Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

doi: 10.18632/oncotarget.7273

Figure Lengend Snippet: ( A , B ) The gap-junction inhibitor CBX inhibits transfer of 22bpCy3 from astrocytes to PC14 cells. PC14 cells (CD340 positive) containing 22bpCy3 were assayed by FACS analysis (see Materials and Methods section). A. Representative dot plots of the results of FACS analysis after the different treatments, depicting total live-cell gate records. B. Quantitative results of FACS analysis expressed as MFI of 22bpCy3 recorded in PC14 cells cultured with astrocytes-22bpCy3 that were treated with CBX (50 μM/150 μM) or untreated (control). CBX inhibited the transfer of endogenous sRNA from the astrocytes to the PC14 cells. C−F. Levels of mmu-miR-16* ( C ), mmu-miR-709 ( D ), mmu-miR-1195 ( E ) and endo-siRNA-1196 ( F ) were measured in PC14-GFP cells by qPCR (see Materials and Methods section). Results are presented as means ± SEM. Analysis (One-way ANOVA revealed significant differences in MFI of 22bpCy3 between the groups: P < 0.05. Post-hoc analysis by Fisher's LSD yielded * P < 0.05, **** P < 0.0001; n = 4.

Article Snippet: The cells were incubated at 37°C in a humidified 5% CO 2 incubator for 6 h, harvested, and stained for 15 min at RT with APC anti-human CD340 Ab to label PC14 cells and with anti-mouse GLAST-PE conjugated antibody (Miltenyi Biotec) to label astrocytes.

Techniques: Cell Culture, Control