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: bioRxiv

Article Title: YTHDC1 m6A-dependent and m6A-independent functions converge to preserve DNA damage response

doi: 10.1101/2024.03.25.586632

Figure Lengend Snippet: A) Schematic representation of p21 endogenously tagged reporter system in A549 cell line. B) Overview of the CRISPR-screening design. A library containing 1,828 guide RNAs was cloned into a lentiviral system and used to infect A549 reporter cells. After 4 days of infection, cells were exposed to Nutlin-3a to allow for p53 protein stabilization and subsequent reporter gene activation. Cells were sorted by FACS based on the level of reporter gene activation. C) Scatter plot of Log2 Fold Change for sgRNA enrichment in p53-enhanced-response (Y-axis) and p53-attenuated-response (X-axis) populations. The three top candidates for each population are labelled and highlighted in the plot. D) Functional annotation of the top 50 candidates for p53-enhanced and p53-attenuated populations based on their molecular activity. E) In vitro validation of the top candidate for p53-enhanced and p53-attenuated populations. Each gene was independently silenced by siRNA. After silencing, mVenus signal was taken as readout of p21-reporter gene activation. Statistical analysis was performed by paired two-tailed Student’s t-test KD versus scramble (SCR)control. F) Pearson correlation score between p53-downstream-effector misregulation (p53 pathway) compared to the expressionof the 407 CRISPR-Screening candidates in Lung carcinoma patients from TCGA (LUAD + LUSC patients) with p53 wild-type genotype versus healthy patients. Data information: All data are shown are representative of at least three independent experiments. Data are presented as mean ± s.d. ns, not significant (P ≥ 0.05), *(P ≤ 0.05), ** (P ≤ 0.01), paired two-tailed student’st-test was performed in E.

Article Snippet: We further modified p21-reporter cells to stably express SpCas9 endonuclease , and transduced these cells with human lentiviral-based CRISPR pooled library in CRISPseq-BFP-backbone vector (Addgene).

Techniques: CRISPR, Clone Assay, Infection, Activation Assay, Functional Assay, Activity Assay, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: YTHDC1 m6A-dependent and m6A-independent functions converge to preserve DNA damage response

doi: 10.1101/2024.03.25.586632

Figure Lengend Snippet: A) Representative Immunoblot of p53, with GAPDH as loading control. Cells were treated with Nutlin-3a or untreated as negative control. B) Nutlin-3a dose response treatment. P21-Reporter cells were treated with increasing doses of Nutlin-3a or untreated as negative control. After treatment, reporter gene activation was measured by flow cytometry. The percentage of cells showing reporter gene activation was calculated and presented in the plot of the signal distribution. C) Representative immunoblot of spCas9, with a-tubulin as loading control. Cells were transduced with a lentivirus carrying spCas9 or an empty vector as negative control. D) Flow cytometry quantification of P21-Reporter gene activation and CRISPR library infection for the two replicates used in the screening. E) Scatter plot of log2 fold change for sgRNA enrichment in p53-enhanced and p53-attenuated populations. Each individual sgRNA for the three top candidates for each population is labelled and highlighted in the plot. Non-targeting sgRNAs used as negative controls in black. F) Scatter plot of log2 fold change for gene enrichment in p53-enhanced and p53-attenuated populations. Previously reported negative or positive regulators of p53 activity are labelled in the plot in red and green, respectively. Functional annotation of the top 50 candidates for p53-enhanced and p53-attenuated populations based on theirmolecular substrate. H) RNA level quantification of mature mRNA by RT-qPCR for YTHDC1 and ASH2L . Cells were transfectedwith two independent siRNA against YTHDC1 (DC1-1 and -2), two independent siRNA against ASH2L (ASH2L-1 and -2) or Scramble (SCR) as negative control. Data information: All data are shown are representative of at least three independent experiments, except for C. Data are presented as mean ± s.d. *** (P ≤ 0.001), **** (P ≤ 0.0001), paired two-tailed student’s t-testwas performed in H.

Article Snippet: We further modified p21-reporter cells to stably express SpCas9 endonuclease , and transduced these cells with human lentiviral-based CRISPR pooled library in CRISPseq-BFP-backbone vector (Addgene).

Techniques: Western Blot, Negative Control, Activation Assay, Flow Cytometry, Transduction, Plasmid Preparation, CRISPR, Infection, Activity Assay, Functional Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 1. A genome-wide CRISPR-Cas9 genetic screen identifies an essential requirement for CRAMP1 and histone H1.4 in PRC2-mediated reporter repression (A) Schematic representation of GFP reporter repression by the PRC2 complex. (B) The GFP reporter is derepressed upon CRISPR-Cas9-mediated gene disruption of any of the three core PRC2 subunits, as assayed by flow cytometry. (C) A genome-wide CRISPR-Cas9 screen to identify factors required for PRC2 function. Following Cas9 expression in KBM-7 cells harboring the PRC2-sensitive GFP reporter, genome-wide mutagenesis was carried out with the Sabatini/Lander single guide RNA (sgRNA) library, 36 and GFP + cells isolated through two sequential rounds of FACS. ‘‘Significance’’ on the y axis represents the negative log of the ‘‘pos|score’’ metric reported by Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK). 37

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: Genome Wide, CRISPR, Disruption, Flow Cytometry, Expressing, Mutagenesis, Isolation, Knock-Out

Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 5. Linker histones are not enriched at regions marked by H3K9me3 (A–D) Lack of linker histone enrichment at H3K9me3-marked genomic regions. (A) Tornado plots depicting linker histone CUT&Tag signal across H3K9me3 peaks from the ENCODE project; average signal intensity is shown in (B). (C) Heatmap depicting the lack of correlation between linker histone occupancy and H3K9me3. Cells are annotated with pairwise Spearman correlation coefficients. An example locus is shown in (D). (E) CUT&Tag faithfully profiles H3K9me3. Example loci comparing CUT&Tag versus H3K9me3 ChIP-seq data (ENCODE) are shown. (F and G) Linker histone insufficiency does not impair H3K9me3-dependent LINE-1 silencing by the HUSH complex. (F) Schematic representation of the dual- color reporter cell line designed to monitor both H3K9me3-dependent repression by the HUSH complex and linker histone-mediated PRC2-reporter repression. (G) HUSH-mediated LINE-1 silencing is unaffected upon CRAMP1 depletion. The indicated CRISPR sgRNAs were expressed in the dual-color reporter cell line, and GFP and iRFP fluorescence assayed by flow cytometry. See also Figure S5 and Table S2.

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: ChIP-sequencing, CRISPR, Fluorescence, Flow Cytometry

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