gerardo ferbeyre  (Addgene inc)

Journal: BMC Biology

Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

doi: 10.1186/s12915-024-01879-0

Figure Lengend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

Article Snippet: On the next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions.

Techniques: Expressing, Western Blot, Clone Assay, Comparison, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test

Journal: BMC Biology

Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

doi: 10.1186/s12915-024-01879-0

Figure Lengend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

Article Snippet: On the next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions.

Techniques: Expressing, Western Blot, Clone Assay, Comparison, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

doi: 10.1186/s12964-024-01570-5

Figure Lengend Snippet: miR-6794-5p directly inhibits the expression of SOCS1. A Venn diagram indicated target candidate genes of miR-6794-5p using TargetScan and miRWalk, which are miRNA target prediction sites. B, C After overexpression of the miR-6794-5p mimic in U251 ( B ) and A549 ( C ) cells, the mRNA expression of each of the candidate genes was confirmed by qRT-PCR. The values were normalized to GAPDH. D After miR-6794-5p was overexpressed in both cells, the level of SOCS1 protein was confirmed by Western blot analysis. β-actin was used for normalization. The experiment was repeated with triplicates and representative Western blotting images are shown. E, F Dual luciferase activity was examined after A549 cells were co-transfected with wild-type (WT) or mutant (Mut) vectors of the SOCS1 3’UTR in the presence or absence of the miR-6794-5p mimic, respectively. G Ago2-RNA immunoprecipitation (Ago2-IP) assay was performed in negative control (NC) or miR-6794-5p overexpressed A549 cells, and SOCS1 enrichment was confirmed by qRT-PCR. The values were normalized to GAPDH. H The mRNA expression of SOCS1 in plasma of normal and patients with lung cancer (normal, n = 23; lung cancer, n = 23) was analyzed by qRT-PCR. I Kaplan-Meier plots were used to compare survival rates between normal groups and lung adenocarcinoma patients. All data are presented as the mean ± S.D. after triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001. Student’s t-test

Article Snippet: The pLPC-flag-SOCS1 vector was provided by Addgene (Plasmid #129514).

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Transfection, Mutagenesis, Immunoprecipitation, Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

doi: 10.1186/s12964-024-01570-5

Figure Lengend Snippet: miR-6794-5p promotes EMT, cell mobility, invasiveness, and stemness maintenance by suppressing SOCS1. A - D U251 and A549 cells were co-transfected with miR-6794-5p or SOCS1 overexpressing vectors. A The expression of EMT and stemness marker proteins in the indicated cells was confirmed by Western blot analysis. β-actin was used for normalization. The experiment was repeated with triplicates and representative Western blotting images are shown. Wound healing ( B ), matrigel coating invasion ( C ), and sphere formation assays ( D ) were performed to confirm cell mobility, invasiveness, and stemness maintenance in the indicated cells. Scale bar is 200 μm. The data are presented as the mean ± S.D. after triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001. One-way ANOVA followed by bonferroni comparison test

Article Snippet: The pLPC-flag-SOCS1 vector was provided by Addgene (Plasmid #129514).

Techniques: Transfection, Expressing, Marker, Western Blot, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

doi: 10.1186/s12964-024-01570-5

Figure Lengend Snippet: miR-6794-5p induces macrophage M2 polarization via JAK1/STAT3 pathway. A Exosomes were isolated from the conditioned media of U251 cells overexpressing Bcl-w. After treatment of THP-1-derived macrophages with exosomes, the mRNA levels of CD163, CD206, and CD11b were measured by qRT-PCR analysis . B, C After THP-1-derived macrophages were treated with conditioned media from U251 and A549 cells transfected with miR-6794-5p mimics, respectively, mRNA levels of macrophage M2 markers (CD163, CD206, and CD11b) were measured by qRT-PCR analysis. D, E mRNA ( D ) or protein ( E ) levels of macrophage M2 markers (CD163, CD206, and CD11b) in THP-1-derived macrophages overexpressing the miR-6794-5p mimic were verified by qRT-PCR or Western blot analysis, respectively. F Using the TCGA dataset, expression of CD206 was compared with normal ( n = 5) and GBM patients ( n = 167) and displayed as box and whisker plots. G The expression level of CD206 in the lung tissues at each stage of lymph node metastasis of patients with lung cancer was confirmed by IHC. H Kaplan-Meier overall survival curves of patients with lung squamous cell carcinoma according to the expression of CD163, CD206 (MRC1), and CD11b (ITGAM). I, J Expressions of p-STAT3, STAT3, p-JAK1, JAK1, and SOCS1 in miR-6794-5p-overexpressed ( I ) or SOCS1-knockdown ( J ) THP-1-derived macrophages were shown by Western blot analysis. K Expression of STAT3 protein after transfection with STAT3 against siRNA in macrophages overexpressing miR-6794-5p (left). Expression mRNA levels of macrophage M2 markers (CD163, CD206, and CD11b) and IL-10 were checked in the indicated cells (right). L After overexpressing empty vector, miR-6794-5p and miR-6794-5p + SOCS1 in THP-1-derived macrophages, the expression of SOCS1 protein was confirmed by Western blot analysis (left), and the mRNA expression levels of macrophage M2 markers (CD163, CD206, and CD11b) were confirmed by qRT-PCR (right) in indicated cells. β-actin was used for normalization in Western blot analysis. The values were normalized to GAPDH in qRT-PCR. The experiment was repeated with triplicates and representative Western blotting images are shown. The data are presented as the mean ± S.D. after triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001. A - D Student’s t-test. K , L One-way ANOVA followed by bonferroni comparison test

Article Snippet: The pLPC-flag-SOCS1 vector was provided by Addgene (Plasmid #129514).

Techniques: Isolation, Derivative Assay, Quantitative RT-PCR, Transfection, Western Blot, Expressing, Whisker Assay, Plasmid Preparation, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

doi: 10.1186/s12964-024-01570-5

Figure Lengend Snippet: miR-6794-5p increases metastasis by inducing M2 polarization in vivo. A - B C57BL/6 mice were injected via tail vein with negative control (NC), miR-6794-5p, or miR-6794-5p + SOCS1 overexpressing LLC1 cells ( n = 5; 5 X 10 5 cells/mouse). Lung tissue was harvested by sacrifice at 4 weeks after cell injection. A Lung tissue images of each group were subjected to H&E and IHC staining with anti-SOCS1. Scale bar is 100 μm. B IHC staining was performed with anti-CD206 using the lung tissues of each group. Scale bars are 100 μm (top) and 50 μm (bottom). C - F Negative control (NC) and miR-6794-5p overexpressed LLC1 cells were subcutaneously injected into the right flank of C57BL/6 mice ( n = 4; 2 X 10 5 cells/mouse). After tumorigenesis, the expression levels of M1 macrophages, M2 macrophages and activated CD8+ T cells in the tumor tissues of the two groups were analyzed by representative flow cytometry. C, D Respective percentages of M1 macrophages (CD45+ F4/80+ CD11b + MHCII+ CD206- cells) and M2 macrophages (CD45+ F4/80+ CD11b + MHCII- CD206+ cells) isolated from tumor tissues developed in negative control (NC) and miR-6794-5p overexpressing mice were analyzed by flow cytometry. E The M1/M2 ratio was calculated based on the percentages of M1 and M2 macrophages analyzed by flow cytometry. F The proportion of activated CD8+ T cells (CD45+ CD8+ CD3+ CD25+ cells) isolated from tumor tissue developed in negative control (NC) and miR-6794-5p overexpressing mice was analyzed by flow cytometry. G Schematic diagram illustrating the mechanism of exosomal miR-6794-5p secreted from tumor cells on surrounding macrophages

Article Snippet: The pLPC-flag-SOCS1 vector was provided by Addgene (Plasmid #129514).

Techniques: In Vivo, Injection, Negative Control, Immunohistochemistry, Expressing, Flow Cytometry, Isolation