Journal: bioRxiv

Article Title: Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

doi: 10.1101/435776

Figure Lengend Snippet: ( A ) Schematic of S1PR1 modulator screening system Four lentiviral vectors were transduced into U2OS cell line to enable gene activation by SAM and monitoring S1PR1 activation by TANGO system. The cells introduced with SAM sgRNA library were starved with 0.5% charcoal treated FBS, then the Venus-positive population was sorted and next-gen sequence (NGS) analysis was carried out to identify the enriched SAM sgRNA sequences. ( B ) Scatter plot showing enrichment of sgRNAs after sorting. Most sgRNAs are equally distributed in the pre-sort sample (closed gray circles) while after sorting a small fraction of sgRNAs (2,770 out of 70,290 sgRNAs) were enriched and others were not detected (open blue circles). The y-axis shows the NGS reads of sgRNAs. ( C ) Identification of top candidate genes using the MAGeCK method . The names of top ten candidate genes are indicated.

Article Snippet: The single clones were isolated from antibiotics resistant cells by limiting dilution, then introduced with the SAM sgRNA library (a gift from Feng Zhang, Addgene #1000000057) at a low multiplicity of infection.

Techniques: Activation Assay, Sequencing

Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: Sorting of miR-200c from cells to exosomes via Rab1A in breast cancer. ( A ) Dig-conjugated miR-200c and let-7a complex pulled down from whole cell extracts of 4T1 lung metastasis using anti-digoxin beads and analyzed by electrophoresis followed by Coomassie blue staining. ( B ) MALDI-TOF-MS analysis of tryptic peptides from the band indicated by red arrows in ( A ). ( C ) Western blot analysis of Rab1A proteins from before (top panel) and after anti-Dig Alpha Donor beads pull-down (bottom panel) in 4T1 lung metastasis lysate. ( D ) ChIP analysis of the interaction between Rab1A and miR-200c using anti-Rab1A antibody pull-down exosomal RNAs and access the miR-200c with miRNA qPCR. ( E ) SPR analysis of the interaction between Rab1A recombinant protein and biotinylated (Bio-) miR-200c or miRNA scramble control covalently immobilized onto the streptavidin-coated sensor chip. ( F ) Rab1A knockout (KO) by transfection of Rab1A CRISPR/Cas9 plasmid with and without miR-200c inhibitor in 4T1 cells. qPCR analysis of miR-200c in exosome and donor cell 3 days after transfection. ( G ) Representative Western blot analysis. ( H ) Quantification of cell number after 48 h culture in 24-well plate. **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: The role of Rab1A on the miR-200c distribution in breast cancer cells was further analyzed by knocking out (KO) the Rab1A gene by transfection of Rab1A CRISPR/Cas9 KO plasmid (Santa Cruz, sc-422550) followed by treatment with or without miR-200c inhibitor in 4T1 cells.

Techniques: Electrophoresis, Staining, Western Blot, Recombinant, Control, Knock-Out, Transfection, CRISPR, Plasmid Preparation

Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: Increased miR-200c trafficking from cell to exosomes promotes breast cancer lung metastasis. ( A ) Representative IHC analysis of Rab1A level in Tri + (n=28) or Tri − (n=25) breast cancer tumor (T) specimens and adjacent normal tissues ( A ) (n=20). Scale bars, 50 μm. ( B ) Quantification of PD-L1 + cell ratio in the specimens. ( C ) Representative immunoblot analysis of Rab1A in the tumor tissues. ( D ) Analysis of miR-200c in lung metastasis using qPCR. ( E ) Representative 4T1 primary breast tumor (top left) and lung (bottom left) with metastatic nodules from tumor-bearing mice (n=5) at 28 days injected with 4T1 cells with Rab1A activation particles via intravenous injection (50 mg/kg, body weight, every other day). ( F ) Quantification of primary tumor volumes (left panel) and metastasis nodule number (>1 μm) (right panel). The tumor size was assessed by the following formula: volume = (width) 2 × length/2. ( G ) Representative immunoblots of Rab1A in tumors (left panel) and qPCR analysis of miR-200c in tumor exosomes (right panel). ( H ) Survival rate of mice in ( E ). *P < 0.05 and **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: The role of Rab1A on the miR-200c distribution in breast cancer cells was further analyzed by knocking out (KO) the Rab1A gene by transfection of Rab1A CRISPR/Cas9 KO plasmid (Santa Cruz, sc-422550) followed by treatment with or without miR-200c inhibitor in 4T1 cells.

Techniques: Western Blot, Injection, Activation Assay

Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: Inhibition of miR-200c sorting protein interferes with breast cancer lung metastasis. ( A ) Survival rate of 4T1 tumor-bearing mice (n=8) treated with different doses of anti-Rab1A antibody or normal lgG control via intravenous injection. ( B ) Representative 4T1 breast primary tumor (top panel) and lung (middle panel) with metastatic nodules from tumor-bearing mice at 28 days after intravenous injection of 4T1 cells with anti-Rab1A antibody (10 mg/kg, body weight, every other day). Quantification of primary tumor volume (bottom left) and metastasis nodule number (>1 μm) (bottom right). ( C ) qPCR analysis of miR-200c in tumor tissues and exosomes. ( D ) Survival rate of mice. *P < 0.05 and **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: The role of Rab1A on the miR-200c distribution in breast cancer cells was further analyzed by knocking out (KO) the Rab1A gene by transfection of Rab1A CRISPR/Cas9 KO plasmid (Santa Cruz, sc-422550) followed by treatment with or without miR-200c inhibitor in 4T1 cells.

Techniques: Inhibition, Control, Injection

Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: 4T1 exosomes inhibit the immune response of immune cells in the lung. ( A ) 4T1-derived exosomes labeled with fluorescence dye PKH16 (Exo/PKH26) and administered to mice by intravenous injection. Immunofluorescence analysis of Exo/PKH26 and F4/80. Cell nuclei stained with DAPI. Scale bars, 50 μm. ( B ) 4T1 tumor-bearing mice (n=8) treated with 4T1-derived exosomes at 50 mg/kg for 3 days via intravenous injection. Concentration of IFNγ and IL-1β in monocytes of breast cancer lung metastasis assessed by flow cytometry. Numbers above the box indicate percent cells. ( C ) 4T1 tumor-bearing mice (n=8) treated with different doses of anti-Rab1A antibody via intravenous injection. Flow cytometry analysis of exosome uptake efficacy by monocytes from lungs. ( D ) Analysis of IFNγ in the lung by ELISA. ( E ) Analysis of IL-1β in the lung by ELISA. *P < 0.05 and **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: The role of Rab1A on the miR-200c distribution in breast cancer cells was further analyzed by knocking out (KO) the Rab1A gene by transfection of Rab1A CRISPR/Cas9 KO plasmid (Santa Cruz, sc-422550) followed by treatment with or without miR-200c inhibitor in 4T1 cells.

Techniques: Derivative Assay, Labeling, Fluorescence, Injection, Immunofluorescence, Staining, Concentration Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Effects of scutellarein on the combination of CDC4 and RAGE proteins. (A) Western blot analysis was performed to investigate the effects of scutellarein (40 µ M) on the expression of ubiquitin-associated proteins, including CDC4, RCHY1, MDM2, CBL and Smurf1 in SW480 cells. (B) An IP assay was performed to detect the effects of scutellarein on the combination of CDC4 and RAGE in SW480 cells. (C) Representative data presenting the levels of ubiquitin-associated proteins (CDC4, RCHY1, MDM2, CBL and Smurf1) in T84 cells treated with scutellarein. (D) An IP assay was used to assess the effects of scutellarein on the combination of CDC4 and RAGE proteins in T84 cells. n=3. * P<0.05 vs. the control group. RAGE, receptor for advanced glycation end products; CDC4, cell division control protein 4; IP, immunoprecipitation; RCHY1, ring finger and CHY zinc finger domain containing 1; MDM2, MDM2 proto-oncogene; CBL, Cbl proto-oncogene; Smurf1, SMAD specific ubiquitin protein ligase 1.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Control, Immunoprecipitation

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Effects of CDC4/RAGE on the proliferation and apoptosis of SW480 and T84 cells. Following 24 h of cell transfection with OE-NC or OE-CDC4, (A) SW480 and (B) T84 cells were treated with CHX (100 µ g/ml) for 1, 2, 4, 8 or 24 h, and then a western blot assay was performed to detect the protein levels of RAGE. (C) A western blot assay was performed to investigate the ubiquitination of RAGE subsequent to SW480 and T84 cells being transfected with OE-CDC4 or OE-NC. Cell proliferation of (D) SW480 and (E) T84 cells was detected using MTT analysis once the cells were transfected with OE-CDC4, OE-CDC4+OE-PAGE or their negative control vector. (F) Effects of CDC4 and RAGE overexpression on the apoptosis of SW480 and T84 cells were analyzed using flow cytometry. n=3. * P<0.05 vs. control group; # P<0.05 vs. the OE-CDC4 group. RAGE, receptor for advanced glycation end products; CDC4, cell division control protein 4; OE, overexpression; NC, negative control; CHX, cycloheximide.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Negative Control, Plasmid Preparation, Over Expression, Flow Cytometry, Control

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Scutellarein treatment decreased RAGE expression and induced cell apoptosis via increasing CDC4 expression. (A) Reverse transcription-quantitative PCR and (B) western blot assays were used to determine the knockdown efficiency of sh-CDC4 in SW480 and T84 cells. * P<0.05 vs. the sh-NC group. Next, SW480 and T84 cells were divided into three groups (control, scutellarein and scutellarein+sh-CDC4), and then submitted to the following assays. (C) Expression of RAGE was detected using a western blot assay. Protein stability of RAGE was detected by using the CHX reagent together with a western blot assay in (D) SW480 and (E) T84 cells. Levels of cleaved caspase3/7 and total caspase3/7 were determined using a western blot assay in (F) SW480 and (G) T84 cells. * P<0.05 vs. the control group; # P<0.05 vs. the scutellarein group. RAGE, receptor for advanced glycation end products; CDC4, cell division control protein 4; sh-, short hairpin RNA; NC, negative control; CHX, cycloheximide.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Control, shRNA, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Effects of scutellarein/CDC4 on the tumor growth in a mice xenograft model with a T84 cell injection. (A) Images of tumors removed from the mice treated with scutellarein and scutellarein+sh-CDC4. Bar graph represents the relative weight of the solid tumor types. (B) Immunohistochemical staining was used to detect the expression of Ki-67 in the tumor tissues derived from different groups. (C) Hematoxylin and eosin staining was used to assess the pathological alteration of the tumor tissues derived from different groups. Data are presented as the mean ± standard error of the mean. n=10. * P<0.05 vs. the control group; # P<0.05 vs. the scutellarein group. CDC4, cell division control protein 4; sh-, short hairpin RNA.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Injection, Immunohistochemical staining, Staining, Expressing, Derivative Assay, Control, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Schematic diagram of the anti-tumor function of scutellarein in colon cancer. Scutellarein treatment reduced the expression of RAGE via the CDC4-mediated ubiquitination degradation pathway, which then inhibited cell proliferation, tumorigenesis and induced cell apoptosis in colon cancer. RAGE, receptor for advanced glycation end products; CDC4, cell division control protein 4.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Ubiquitin Proteomics, Control

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: A . Slides without primary antibody served as the negative control in GC tissues, B, E . Typical immunohistologic features with Rap1GAP expression in para-carcinoma and GC tissues, the Rap1GAP staining localized predominantly in the cytoplasm. C, F . Typical immunohistologic features with E-cadherin expression in para-carcinoma and GC tissues. D . Immunostaining for MMP2 was performed in GC; Magnifications, ×200.

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Negative Control, Expressing, Staining, Immunostaining

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Differences in Rap1GAP, E-cadherin and MMP2 between the cancer tissues and para-carcinoma tissues

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Rap1GAP and E-cadherin protein in GC cells were notably lower expression compared to 293T cells by western blotting. Conversely, MMP2 was upregulated in GC cells (*P < 0.05 and **P < 0.01)

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Correlations between the clinicopathologic variables with Rap1GAP, E-cadherin and MMP2

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Association between Rap1GAP with E-cadherin and MMP2

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: The cumulative overall survival differences between the patients with high and low levels of protein expression. The P value was obtained using the log-rank test of the difference. A . Rap1GAP; B . E-cadherin; C . MMP2.

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Expressing

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Univariate Analysis for Overall Survival

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Multivariate Cox Proportional Hazards Analysis for Overall Survival

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: The GC cells were grown and transfected with the negative control (NC) or Rap1GAP CRISPR Activation Plasmid. Overexpression Rap1GAP improved the expression of E-cadherin (A, B) and, inversely, suppressed the expression of MMP2 (A, B) compared to NC group in mRNA level and protein level. (*P < 0.05 and **P < 0.01).

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Transfection, Negative Control, CRISPR, Activation Assay, Plasmid Preparation, Over Expression, Expressing

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Overexpression Rap1GAP repressed the migration A . and invasion B . capacity of GC cells C . The graph shows the quantified data of the assay, migration (left) and invasion (right) (**P < 0.01).

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Over Expression, Migration

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Temporal expression of endogenous MC1R and Nurr1 in the ipsilateral brain hemisphere post-HI. (a) Representative Western blot bands of the time course. (b) Western blot data showed that the endogenous expression levels of MC1R significantly increased from 12 h reaching peak at 48 h post-HI. (c) Nurr1 expression levels significantly increased over time, reached highest at the 48 h post-HI. n = 6 per group. Data were represented as mean ± SD. ∗ p < 0.05 versus sham, # p < 0.05 versus 6 h HI, and & p < 0.05 versus 48 h HI; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Expressing, Western Blot, Hi-C

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Immunofluorescence staining showed MC1R and Nurr1 colocalization with neurons in the ipsilateral brain hemisphere at 48 h post-HI. Immunofluorescence staining showed that MC1R (a) and Nurr1 (b) expressions on neurons were seen to be higher in the vehicle-treated pup rats compared to the sham group, and a higher expression of MC1R (a) and Nurr1 (b) on neurons after BMS-470359 treatment compared with vehicle. Merge showed the colocalization of MC1R and Nurr1 on neurons. n = 2 per group. Neurons were stained red. MC1R and Nurr1 were stained green. DAPI was stained blue. Scale bar = 100 μ m.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Immunofluorescence, Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Effects of BMS-470539 treatment on neuronal apoptosis at 48 h post-HI, and the knockout efficiency of MC1R or Nurr1 knockout CRISPR in naive and HI rats. (a, b) Representative microphotographs and quantitative analysis of C-Cas 3-positive neurons in the ipsilateral hemisphere at 48 h post-HI. The number of C-Cas 3-positive neurons significantly increased in the vehicle group compared with the sham group and BMS-470539 treatment group. n = 4 per group. C-Cas 3 was green. Neurons were red. Blue was for DAPI. Scale bar = 100 μ m. (c, d) Representative Western blot bands and quantitative analysis of MC1R and Nurr1 protein levels in the ipsilateral hemisphere at 48 h post-HI. The expression of MC1R or Nurr1 was significantly reduced by MC1R or Nurr1 knockout CRISPR in naive and HI rats. n = 4 per group (data were represented as mean ± SD; ∗ p < 0.05 versus sham; # p <0.05 versus HI+vehicle; @ p < 0.05 versus naive+control CRISPR, & p < 0.05 versus HI+control CRISPR; one-way ANOVA, Tukey's post hoc test).

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 treatment exerted its antioxidative stress and antiapoptosis effects via MC1R/cAMP/PKA/Nurr1 signaling pathway at 48 h post-HI. (a) Representative Western blot bands. (b–i) Quantification of MC1R, cAMP, p-PKA, Nurr1, 4-HNE, HO-1, Bax, and Bcl-2 expression levels in the ipsilateral hemisphere at 48 h post-HI. BMS-470539 treatment significantly increased the protein levels of MC1R, cAMP, p-PKA, Nurr1, HO-1, and Bcl-2 but significantly decreased the expression of 4-HNE and Bax compared to the HI+vehicle group. Knockout of MC1R or Nurr1 with CRISPR interventions significantly abolished such effects of BMS-470539. n = 6 per group. Data was represented as mean ± SD. ∗ p < 0.05 versus sham, # p < 0.05 versus HI+vehicle, @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Western Blot, Expressing, Knock-Out, CRISPR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 administration reduced neuronal degeneration at 48 h post-HI, which was reversed by MC1R or Nurr1 knockout CRISPR. (a, b) Representative microphotographs and quantitative analysis of FJC-positive neurons in the ipsilateral hemisphere at 48 h post-HI. n = 6 per group. FJC was green. Blue was for DAPI. Scale bar = 100 μ m. Data was represented as mean ± SD. ∗ p < 0.05 versus sham; # p < 0.05 versus HI+vehicle; @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 administration suppressed oxidative stress at 48 h post-HI, which was abolished by MC1R or Nurr1 knockout CRISPR. (a, b) Representative microphotographs of MitoSox (red) and 8-OHdG (green) staining in the ipsilateral hemisphere at 48 h post-HI. Blue was for DAPI. (c, d) Quantitative analysis of MitoSox- and 8-OHdG-positive cells. n = 6 per group. Scale bar = 100 μ m. Data was represented as mean ± SD. ∗ p < 0.05 versus sham; # p < 0.05 versus HI+vehicle; @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Staining, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: Clinical and molecular features of patients with tumor molecular profiling on biopsies obtained upon resistance to ALK inhibitors in the MATCH-R study.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Biomarker Discovery, Sequencing, RNA Sequencing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: NF2 loss of function mediates resistance to lorlatinib. A, Clinical course of patient MR135 and mutational profile of samples obtained on lorlatinib progression (PD, progressive Disease). B, Cell survival assay assessed with Cell Titer Glo of MR135 lorlatinib resistant cells from biopsy 1 (MR135-R1) treated for 7 days with the indicated concentrations of lorlatinib and vistusertib (AZD2014) alone or in combination. C, Immunoblot analysis from cell lysates of MR135-R1 treated for 24hs with the specified doses of lorlatinib, vistusertib (AZD2014) and ponatinib alone or in combination using indicated antibodies. D, Athymic nude mice bearing MR135-R2 PDX were administered lorlatinib or vistusertib 20 mg/kg orally. Tumor volumes, mean ±SD (n =8); (*** p < 0.001). E, Cell lysates from H3122 parental and H3122 cells with NF2 heterozygous deletions or homozygous deletions, generated by CRISPR-CAS9 gene editing, were immunoblotted to detect merlin expression. H3122 cells with bi-allelic NF2 knock-out lacked merlin expression. F, Cell survival assay of H3122 parental and H3122 NF2 knock-out (NF2 KO) cells treated with lorlatinib for 7 days. Cell survival was assessed by Cell Titer Glo. G, Cell proliferation assay of H3122 parental and H3122 NF2 KO cells untreated and treated with lorlatinib measured at baseline, day 2, day 5 and day 7. Cell viability was assessed with Cell Titer Glo. H, Caspase 3/7 activation (Caspase 3/7-Glo assay) relative to the number of live cells simultaneously assessed in the cell proliferation assay previously described. I, H3122 parental and NF2 KO cells were treated with the indicated doses of lorlatinib for 24hs. Cell lysates were immunoblotted to detect the selected proteins.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Clonogenic Cell Survival Assay, Western Blot, Generated, CRISPR, Expressing, Knock-Out, Proliferation Assay, Activation Assay, Glo Assay