g3bp1 gfp  (Addgene inc)

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: HIF-1α associates with PRMT6 and regulates its transcription. ( A ) Correlation analysis of HIF-1α and PRMT6 expression in gliomas using mRNA sequencing data from TCGA and CGGA databases. ( B ) Time-course analysis of HIF-1α, PRMT6, and G3BP1 protein expression in LN229 and U251 cells under hypoxic conditions. ( C ) HIF-1α, PRMT6 and G3BP1 protein expression in LN229 and U251 cells treated with CoCl2 concentration gradient. ( D ) Immunofluorescence analysis of HIF-1α (red) and PRMT6 (green) in HIF-1α-knockdown cells under normoxia or hypoxia conditions. Scale bar = 100 μm. ( E ) HIF-1α, PRMT6 and G3BP1 protein expression in HIF-1α-knockdown cells under normoxia or hypoxia conditions. ( F ) HIF-1α and PRMT6 mRNA expression in knockdown cells under normoxic or hypoxic conditions. ( G ) Five predicted HIF-1α binding sites in PRMT6 promoter region. ( H ) ChIP-qPCR analysis of HIF-1α binding at predicted sites in U251 and LN229 cells, showing strongest binding at HRE3. ( I ) Dual-luciferase reporter assay of HRE3-driven PRMT6 promoter activity in HIF-1α-knockdown U251 and LN229 cells under normoxic or hypoxic conditions. Data in B-F, H, and I are presented as mean ± SD. For B-F, H, and I, n = 3 biologically independent samples. Statistical significance in B-F, H, and I was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: PRMT6 overexpression plasmids and empty vectors were obtained from TsingKe (Beijing, China). shG3BP1, shGABPA, and their respective control plasmids, along with G3BP1 overexpression plasmids, were sourced from GeneChem (China).

Techniques: Expressing, Sequencing, Concentration Assay, Immunofluorescence, Knockdown, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: Transcriptomic analysis identifies G3BP1 as the key mediator of PRMT6-induced temozolomide resistance. ( A ) Comparative TMZ IC50 values between isogenic wild-type and resistant variants of U251 and LN229 cell lines. ( B ) Flow cytometric apoptosis profiles of isogenic wild-type and resistant cell lines treated with 200µM TMZ. ( C ) Quantitative apoptosis ratios in wild-type versus resistant variants following 200µM TMZ treatment. ( D ) Colony formation assay of isogenic cell pairs under TMZ concentration gradients. ( E ) Tumor sphere size comparison between wild-type and resistant variants cultured with 100µM TMZ for 24 h. Scale bar = 500 μm. ( F ) Volcano plot of differential gene expression in PRMT6-knockdown versus control U251 or LN229 cell lines, identifying PRMT6 and G3BP1 as significantly downregulated genes. ( G ) Volcano plot of U251 TMZ-resistant versus wild-type cells, showing PRMT6 and G3BP1 as prominently upregulated genes in resistant variants. ( H ) Venn diagram intersection of differentially expressed genes from PRMT6-knockdown cells and TMZ-resistant variants, highlighting G3BP1 among 23 overlapping genes with strongest PRMT6 correlation. ( I ) Heatmap visualization of the 23 intersecting genes’ expression patterns in resistant versus wild-type cell pairs. ( J ) Western blot analysis of PRMT6 and G3BP1 protein expression in U251 wild-type and its isogenic TMZ-resistant counterpart. ( K ) Immunofluorescence analysis of PRMT6 (green) and G3BP1 (red) in wild-type and resistant variants. Scale bar = 100 μm. Data in A, C, D, E, J, and K are presented as mean ± SD. For A, B, D, E, F, G, J, and K, n = 3 biologically independent samples. Statistical significance in A, C, D, E, J, and K was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as source data files

Article Snippet: PRMT6 overexpression plasmids and empty vectors were obtained from TsingKe (Beijing, China). shG3BP1, shGABPA, and their respective control plasmids, along with G3BP1 overexpression plasmids, were sourced from GeneChem (China).

Techniques: Colony Assay, Concentration Assay, Comparison, Cell Culture, Gene Expression, Knockdown, Control, Expressing, Western Blot, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: PRMT6 interacts with GABPA to transcriptionally regulate G3BP1 expression. ( A ) Correlation analysis of PRMT6 and G3BP1 expression in gliomas using mRNA sequencing data from TCGA and CGGA databases. ( B ) PRMT6 and G3BP1 protein expression in U251 and LN229 cell lines following PRMT6 knockdown. ( C ) PRMT6 and G3BP1 mRNA expression in U251 and LN229 cells lines after PRMT6 knockdown. ( D ) Immunofluorescence analysis of PRMT6 (green) and G3BP1 (red) in PRMT6-knockdown U251 and LN229 cells. Scale bar = 100 μm. ( E ) Venn diagram showing overlap between PRMT6-interacting proteins identified by mass spectrometry in LN229 cells and G3BP1-regulating transcription factors from Cistrome and KnockTF databases. ( F ) AlphaFold3-predicted molecular docking model of PRMT6 and GABPA proteins. ( G ) Co-immunoprecipitation (Co-IP) assay demonstrating PRMT6-GABPA interaction in U251 and LN229 cells. ( H ) Fluorescence co-localization of PRMT6 (green) and GABPA (red). Scale bar = 10 μm. ( I ) Eight predicted GABPA binding sites in the G3BP1 promoter region. ( J ) ChIP-qPCR analysis of PRMT6 and GABPA binding at predicted sites in U251 and LN229 cells, showing strongest binding at HRE5. ( K ) Dual-luciferase reporter assay measuring HRE5-driven G3BP1 promoter activity in GABPA-knockdown versus control U251 and LN229 cells. ( L ) ChIP-qPCR analysis of PRMT6 and GABPA binding to G3BP1 HRE5 in GABPA-knockdown and control cells. ( M ) Dual-luciferase reporter assay in HEK-293T cells under PRMT6 knockout conditions to evaluate luciferase activity in GABPA-knockdown versus control groups. ( N ) G3BP1 protein expression in HEK-293T cells with combined PRMT6 knockdown and GABPA knockdown. Data in B-D, and J-M are presented as mean ± SD. For B-D, G, H, and J-N, n = 3 biologically independent samples. Statistical significance in B-D, and J-M was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: PRMT6 overexpression plasmids and empty vectors were obtained from TsingKe (Beijing, China). shG3BP1, shGABPA, and their respective control plasmids, along with G3BP1 overexpression plasmids, were sourced from GeneChem (China).

Techniques: Expressing, Sequencing, Knockdown, Immunofluorescence, Mass Spectrometry, Co-Immunoprecipitation Assay, Fluorescence, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Activity Assay, Control, Knock-Out

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: G3BP1 is highly expressed in gliomas and contributes to TMZ resistance. ( A ) Pan-cancer analysis of G3BP1 in the TCGA and GTEx databases shows that G3BP1 is generally highly expressed in tumors compared to normal tissues, with a significant difference in glioma. ( B ) Univariate Log-rank test of pan-cancer survival data in the TCGA dataset indicates that G3BP1 is associated with poor prognosis in glioma. ( C ) Comparative analysis of G3BP1 mRNA expression among GBM, LGG, and normal brain tissues using TCGA and GTEx data. ( D ) Kaplan-Meier survival analysis shows the overall survival of glioma patients with high and low G3BP1 expression in the TCGA and CGGA datasets. ( E ) Kaplan-Meier survival analysis of overall survival in glioma patients treated with temozolomide, stratified by high and low G3BP1 expression in the CGGA dataset. ( F ) Verification of G3BP1 protein expression after G3BP1 knockdown in U251 and LN229 cell lines. ( G ) Verification of G3BP1 mRNA expression after G3BP1 knockdown in U251 and LN229 cell lines. ( H ) Decreased TMZ IC50 values in G3BP1-knockdown cells indicate enhanced TMZ sensitivity. ( I ) Flow cytometric apoptosis profiles of G3BP1-deficient U251/LN229 cells following 200µM TMZ exposure. ( J ) Quantitative apoptosis ratios in G3BP1-knockdown cells following 200µM TMZ treatment. Data in A, B, C, G, H and J are presented as mean ± SD. For F-I n = 3 biologically independent samples. Statistical significance in A, C, G, H and J was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. The significance of data in B, D, and E was assessed using the Log-rank test for survival comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: PRMT6 overexpression plasmids and empty vectors were obtained from TsingKe (Beijing, China). shG3BP1, shGABPA, and their respective control plasmids, along with G3BP1 overexpression plasmids, were sourced from GeneChem (China).

Techniques: Expressing, Knockdown

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: Hypoxia promotes TMZ resistance in glioblastoma through the HIF-1α-PRMT6-G3BP1 axis. ( A ) G3BP1 protein expression in LN229 cells with PRMT6 knockdown versus controls under normoxic or hypoxic conditions, demonstrating that hypoxia-mediated regulation of G3BP1 is less pronounced than PRMT6-mediated regulation. ( B ) TMZ IC50 values in PRMT6-knockdown and control LN229 cells under different oxygen conditions. ( C ) Flow cytometric apoptosis profiles of PRMT6-knockdown and control LN229 cells treated with 200µM TMZ under normoxic or hypoxic conditions. ( D ) Quantitative apoptosis ratios in PRMT6-knockdown versus control LN229 cells following 200µM TMZ treatment under normoxic or hypoxic conditions. ( E ) Rescue experiment showing HIF-1α and PRMT6 protein expression in HIF-1α-knockdown LN229 cells with PRMT6 overexpression, indicating partial restoration of HIF-1α knockdown effects. ( F ) TMZ IC50 values in the HIF-1α knockdown/PRMT6 overexpression rescue experiment. ( G ) Flow cytometry apoptosis profiles of 200µM TMZ-induced apoptosis in the HIF-1α knockdown/PRMT6 overexpression rescue model. ( H ) Quantitative apoptosis ratios in the HIF-1α knockdown/PRMT6 overexpression rescue experiment following 200µM TMZ treatment. ( I ) Rescue experiment showing PRMT6 and G3BP1 protein expression in PRMT6-knockdown LN229 cells with G3BP1 overexpression, indicating partial restoration of PRMT6 knockdown effects. ( J ) TMZ IC50 values in the PRMT6 knockdown/G3BP1 overexpression rescue experiment. ( K ) Flow cytometry apoptosis profiles of 200µM TMZ-induced apoptosis in the PRMT6 knockdown/G3BP1 overexpression rescue model. ( L ) Quantitative apoptosis ratios in the PRMT6 knockdown/G3BP1 overexpression rescue experiment following 200µM TMZ treatment. Data in B, D, F, H, J, and L are presented as mean ± SD. For A-C, E-G, I-K n = 3 biologically independent samples. Statistical significance in B, D, F, H, J, and L was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: PRMT6 overexpression plasmids and empty vectors were obtained from TsingKe (Beijing, China). shG3BP1, shGABPA, and their respective control plasmids, along with G3BP1 overexpression plasmids, were sourced from GeneChem (China).

Techniques: Expressing, Knockdown, Control, Over Expression, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: G3BP1 suppresses the translation of BCL2L13 mRNA by sequestering it into stress granules. ( A ) KEGG pathway analysis of PRMT6 expression in TCGA glioma dataset demonstrates PRMT6-mediated suppression of apoptosis pathways. ( B ) GSEA pathway analysis of PRMT6 expression in TCGA glioma dataset confirms PRMT6’s inhibitory effect on apoptotic pathways. ( C ) Heatmap analysis from CPTAC glioma proteomics database showing correlation between stress granule genes (represented by G3BP1) and apoptosis-related genes, with BCL2L13 demonstrating the most significant negative correlation with G3BP1. ( D ) BCL2L13 transcript abundance in the RNA-seq dataset ( GSE138058 ) with or without sodium arsenite stimulation. ( E ) Protein expression of G3BP1 and BCL2L13 in U251 and LN229 wild-type cells during 100µM TMZ treatment and after TMZ withdrawal. ( F ) mRNA expression of G3BP1 and BCL2L13 in U251 and LN229 wild-type cells during 100µM TMZ treatment and after TMZ withdrawal. ( G ) Protein expression of G3BP1 and BCL2L13 in G3BP1-knockdown U251 and LN229 cells following 6 h stimulation with 100µM TMZ. ( H ) mRNA expression of G3BP1 and BCL2L13 in G3BP1-knockdown U251 and LN229 cells following 6 h stimulation with 100µM TMZ. (I) RIP-qPCR analysis showing the enrichment of BCL2L13 mRNA in immunoprecipitates using an anti-G3BP1 antibody in TMZ-treated cells. (J) RNA fluorescence in situ hybridization was performed on wild-type cells using a BCL2L13 mRNA-specific probe under two conditions: untreated and following 6-hour stimulation with 100µM TMZ. Arrows indicate regions of stress granule formation and mRNA accumulation. G3BP1 is shown in green, and BCL2L13 mRNA signals are shown in red. Scale bar = 10 μm. ( K ) RNA fluorescence in situ hybridization with a BCL2L13 mRNA-specific probe was performed in both G3BP1-knockdown and corresponding control cells following TMZ treatment. Arrows indicate regions of stress granule formation and mRNA accumulation. G3BP1 is shown in green, and BCL2L13 mRNA signals are shown in red. Scale bar = 10 μm. ( L ) Schematic diagram of the proposed mechanism: hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma through G3BP1. Data in D-I are presented as mean ± SD. For E-K n = 3 biologically independent samples. Statistical significance in D-I was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: PRMT6 overexpression plasmids and empty vectors were obtained from TsingKe (Beijing, China). shG3BP1, shGABPA, and their respective control plasmids, along with G3BP1 overexpression plasmids, were sourced from GeneChem (China).

Techniques: Expressing, RNA Sequencing, Knockdown, Fluorescence, In Situ Hybridization, Control

Journal: Journal of Translational Medicine

Article Title: Hypoxia-induced PRMT6 expression promotes temozolomide chemoresistance in glioblastoma via G3BP1

doi: 10.1186/s12967-025-07618-5

Figure Lengend Snippet: PRMT6 Promotes Glioblastoma Proliferation and Temozolomide Resistance In Vivo. ( A ) Schematic diagram of the animal experimental protocol. ( B ) The survival curve was generated from an orthotopic glioblastoma mouse model established by intracranial implantation of the following cell variants: (i) PRMT6 shCTRL + Empty Vector + DMSO; (ii) PRMT6 shRNA + Empty Vector + DMSO; (iii) PRMT6 shCTRL + Empty Vector + TMZ; (iv) PRMT6 shRNA + Empty Vector + TMZ; (v) PRMT6 shCTRL + G3BP1 OE + TMZ; and (vi) PRMT6 shRNA + G3BP1 OE + TMZ. ( C ) Representative H&E-stained brain sections showing orthotopic xenografts. Top panels: scale bar = 1.5 mm; Bottom panels: scale bar = 100 μm. ( D ) Representative immunohistochemical staining images of PRMT6, G3BP1 and BCL2L13 in intracranial tumors from nude mice. Scale bar = 100 μm. ( E ) Intracranial tumor volume was measured using brain sections from orthotopic glioblastoma mouse models. ( F ) Effects of PRMT6 and G3BP1 on TMZ-treated subcutaneous tumor growth were assessed (treatment initiation criteria: tumor volume ≈ 50 mm³). The PRMT6 shCTRL + Empty Vector + TMZ and PRMT6 shCTRL + G3BP1 OE + TMZ groups received treatment at day 7, while the PRMT6 shRNA + Empty Vector + TMZ and PRMT6 shRNA + G3BP1 OE + TMZ groups were treated at day 15. ( G ) Comparison of subcutaneous tumor sizes in nude mouse xenograft models. ( H ) Protein expression of PRMT6, G3BP1, and BCL2L13 in intracranial xenograft tissues of nude mice. Data in B, E and F are presented as mean ± SD. For B-G n = 5 biologically independent samples; H n = 3 biologically independent samples. Statistical significance in B, E and F was assessed by one-way ANOVA followed by Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: PRMT6 overexpression plasmids and empty vectors were obtained from TsingKe (Beijing, China). shG3BP1, shGABPA, and their respective control plasmids, along with G3BP1 overexpression plasmids, were sourced from GeneChem (China).

Techniques: In Vivo, Generated, Plasmid Preparation, shRNA, Staining, Immunohistochemical staining, Comparison, Expressing

Journal: Scientific Reports

Article Title: mRNA recruitment by G3BP1 condensates is regulated by Caprin1 but requires G3BP1 binding to mRNA

doi: 10.1038/s41598-025-21066-7

Figure Lengend Snippet: Caprin1 decreases the RNA and G3BP1 concentration thresholds required for the formation of G3BP1-rich condensates.

Article Snippet: Plasmids encoding G3BP1, G3BP1-ΔC, G3BP1-ΔN and G3BP1-ΔIDR2 fused with RFP-tag on N-terminus were constructed by amplification corresponding cDNAs with primers containing appropriate digestion sites and inserted into the mRFP-C1 vector (Addgene).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: mRNA recruitment by G3BP1 condensates is regulated by Caprin1 but requires G3BP1 binding to mRNA

doi: 10.1038/s41598-025-21066-7

Figure Lengend Snippet: G3BP1 and Caprin1 interact in cellular context. Schematic representation of the Microtubule Bench assay. In brief, G3BP1 (bait) fused to microtubule-associated domains of Tau (MBD) and RFP is brought onto microtubules in living cells whereas the presence of a GFP-fused protein partner (prey) on microtubules reveals the interaction by colocalization of the fluorescence signals on microtubules. Top: HeLa cells were co-transfected with the plasmids encoding G3BP1-RFP-MBD and the plasmid expressing the full length Caprin1, YBX1, G3BP2 or EIF4B, all fused to GFP. The scale bar represents 3 μm. Bottom: Scatter plot representing the colocalization level of MBD-fused G3BP1-RFP with one of the eight tested proteins. Each data point represents the average correlation coefficient between fluorescence intensities from red and green channels in one well. The plot shows the data from four independent experiments. Red lines show mean values. Significances between correlation coefficients were obtained using t test; ∗∗∗p < 0.005. Representative images of HeLa cells subjected to oxidative stress via 300 µM sodium arsenite treatment expressing GFP-labeled proteins, with endogenous G3BP1 tracked via specific antibodies, and mRNA tracked via oligo-dT-(Cy5) fluorescent probe. Scale bar: 20 µm. Scatter plot representing the relative protein-GFP enrichment in SGs with (si-G3BP1) or without (si-Neg) decreasing endogenous G3BP1 levels with si-RNA. Each dot corresponds to the average enrichment in all SGs present in the cytoplasm of all cells analyzed per well. Significances between protein enrichment levels were obtained using t test; * ∗p < 0.01, ∗∗∗p < 0.005; ns, not significant. Mean value are below each scatter plot. Same as ( D ) with the RNA enrichment in SGs.

Article Snippet: Plasmids encoding G3BP1, G3BP1-ΔC, G3BP1-ΔN and G3BP1-ΔIDR2 fused with RFP-tag on N-terminus were constructed by amplification corresponding cDNAs with primers containing appropriate digestion sites and inserted into the mRFP-C1 vector (Addgene).

Techniques: Fluorescence, Transfection, Plasmid Preparation, Expressing, Labeling, Protein Enrichment

Journal: Scientific Reports

Article Title: mRNA recruitment by G3BP1 condensates is regulated by Caprin1 but requires G3BP1 binding to mRNA

doi: 10.1038/s41598-025-21066-7

Figure Lengend Snippet: Caprin1 promotes G3BP1 RNA recruitement. Top: scatter plot representing the colocalization level between G3BP1-RFP-MBD brought on MT and Caprin1 or mRNA in the presence of endogenous level of Caprin1 or overexpressed Caprin1-GFP. Each data point represents the average correlation coefficient between fluorescence intensities from RFP and GFP or cyanin5 in one well. Red lines show mean values. Significances between correlation coefficients were obtained using t test; ∗∗∗p < 0.005. Bottom: schematic representation of the MT bench principle and the detection scheme used for endogenous Caprin1 and mRNA. ssDNA mobility shift assay demonstrating the different interaction between Caprin1 or G3BP1 and single stranded nucleic acid. 100 ng of m13 ssDNA were incubated with increasing concentrations of G3BP1 (lanes 2–6) or Caprin1 (lanes 8–12). ssDNA mobility shift assay demonstrating the cooperation between Caprin1 and G3BP1 for binding to single stranded nucleic acids. 100 ng of m13 ssDNA were incubated with increasing concentrations of a mix between G3BP1 and Caprin1 (lane 2–6), Caprin1 alone (lanes 7–11) or G3BP1 alone (lanes 12–16). ssDNA mobility shift assay demonstrating the different integration of Caprin1 into preformed G3BP1-m13 ssDNA complexes (lanes 2–7) or oppositely, of G3BP1 into preformed Caprin1-m13 ssDNA complexes (lanes 8–13). 100 ng of m13 ssDNA were incubated for 1 min with 16 pmol of G3BP1 before the addition of increasing concentration of Caprin1 or with 12 pmol of Caprin1 before the addition of increasing concentration of G3BP1. Left: schematic representation of the mixing/demixing experiment. Two RBPs, as indicated, are confined on the microtubule network (fused to RFP/GFP-MBD) to visualize their mixing/demixing in HeLa cells. Mixing: yellow microtubules. Demixing: red and green microtubules. Middle: scatter plot representing the mixing between G3BP1 and different RBPs, both fused to MBD. Each data point represents the average value obtained in cells analyzed in one well. The mixing score is the value of the determination coefficient R 2 calculated as described in the experimental procedures section. The plot shows the data from eight independent experiments. Red lines show mean values. Significances between protein enrichment levels were obtained using t test; ∗∗∗p < 0.005. Right: representative images for a low (PABPC1L) and a high (G3BP1 and Caprin1) or medium (YBX1) mixing with G3BP1. Top: atomic force microscopy images and zoom-in on specific assemblies of G3BP1/RNA, Caprin1/RNA, and of mixture of the two RBPs with 2luc mRNA. Proteins (20 nM of G3BP1 or 10 nM of Caprin1 or a mixture of 10 nM G3BP1/5 nM Caprin1) were incubated with 2luc mRNA (protein/nucleotide ratio of 1/100) for 5 min before sample deposition and fixation. White arrows pointed to RNA/RBP complexes. Z scale 8 nm. The scale bar represents 400 nm (200 nm in zooms). Bottom: scatter plot representing the area of isolated RBP/RNA assemblies observed on AFM images and comparison with free mRNA. Only areas of RNA or RBP/RNA complexes with a value higher than 200 nm 2 are plotted to discard free proteins from this analysis. Each point corresponds to the same surface analyzed, here 100 μm 2 . The plot gathers the data from two independent experiments. Red lines show mean values. Significances between samples were obtained using t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005.

Article Snippet: Plasmids encoding G3BP1, G3BP1-ΔC, G3BP1-ΔN and G3BP1-ΔIDR2 fused with RFP-tag on N-terminus were constructed by amplification corresponding cDNAs with primers containing appropriate digestion sites and inserted into the mRFP-C1 vector (Addgene).

Techniques: Fluorescence, Mobility Shift, Incubation, Binding Assay, Concentration Assay, Protein Enrichment, Microscopy, Isolation, Comparison

Journal: Scientific Reports

Article Title: mRNA recruitment by G3BP1 condensates is regulated by Caprin1 but requires G3BP1 binding to mRNA

doi: 10.1038/s41598-025-21066-7

Figure Lengend Snippet: MT bench analysis reveals that G3BP1 RNA binding is of fundamental importance in mRNA recruitment by the G3BP1/Caprin1 complex. Schematic representation of the G3BP1 constructs. Right panel: HEK293 cells were co-transfected with Caprin1-GFP or YBX1-GFP and RFP-G3BP1 FL, RFP-G3BP1-ΔC, RFP-G3BP1-ΔN or RFP-G3BP1-ΔIDR2. G3BP1 FL - YBX1 co-transfection was used as a control. Cells were lysated with buffer containing RNAse A and immunoprecipitation was performed using a-GFP antibodies. Left panel: Western blot analysis of cell lysates. The average efficiency of GFP IP was approximately 34% for Caprin1-GFP and 3% for YBX1-GFP. Scatter plot representing the colocalization level of MBD-fused G3BP1-RFP constructs with overexpressed Caprin1-GFP. Each data point represents the average correlation coefficient between fluorescence intensities from red and green channels in one well. The plot shows the data from eight independent experiments. Red lines show mean values. Significances between correlation coefficients were obtained using t test; ∗∗∗p < 0.005; ns: not significant. Scatter plot representing the colocalization level of MBD-fused G3BP1-RFP constructs with mRNA. Each data point represents the average correlation coefficient between fluorescence intensities from red and cyanin5 channels in one well. The plot shows the data from eight independent experiments. Red lines show mean values. Significances between correlation coefficients were obtained using t test; ∗p < 0.05; ∗∗∗p < 0.005; ns: not significant. Representative images of MT in HeLa cells when transfected with the constructions encoding Caprin1-GFP and G3BP1-RFP-MBD for the different G3BP1 truncations and mutations. The scale bar represents 3 μm. Schematic representation of the MT bench experiment where Caprin1 was brought on microtubule and the detection scheme to reveal interaction with overexpressed RFP-G3BP1 constructs and mRNA. Scatter plot representing the colocalization level of MBD-fused Caprin1-GFP with different G3BP1 constructs or RFP alone as a control. Each data point represents the average correlation coefficient between fluorescence intensities from red and cyanin5 channels in one well. The plot shows the data from eight independent experiments. Red lines show mean values. Significances between correlation coefficients were obtained using t test; ∗p < 0.05; ∗∗∗p < 0.005. Same as (G) but the correlation coefficient measures the colocalization between MBD-fused Caprin-GFP and mRNA. Significances between correlation coefficients were obtained using t test: ∗∗∗p < 0.005;.

Article Snippet: Plasmids encoding G3BP1, G3BP1-ΔC, G3BP1-ΔN and G3BP1-ΔIDR2 fused with RFP-tag on N-terminus were constructed by amplification corresponding cDNAs with primers containing appropriate digestion sites and inserted into the mRFP-C1 vector (Addgene).

Techniques: RNA Binding Assay, Construct, Transfection, Cotransfection, Control, Immunoprecipitation, Western Blot, Fluorescence

Journal: Scientific Reports

Article Title: mRNA recruitment by G3BP1 condensates is regulated by Caprin1 but requires G3BP1 binding to mRNA

doi: 10.1038/s41598-025-21066-7

Figure Lengend Snippet: Caprin1 overexpression is not sufficient to restore mRNA recruitment in SG in cells expressing G3BP1-ΔC. Representative images of HeLa cells expressing G3BP1-FL, -ΔN, -ΔC or -ΔIDR2 plasmids. Scale bar: 40 µm. Scatter plot representing the area of mRNA-rich granules in HeLa following the overexpression of different G3BP1 constructs. Each dot corresponds to the average area of granules of all cells of a well expressing the G3BP1 construct. Data were obtained from four independent samples. Granules were detected using cyanin5 fluorescence signal. Significances between granule areas were obtained using t test; ∗∗∗p < 0.005; mean value in red. Same as (B) but the G3BP1 truncation enrichment in mRNA-rich granule is measured. Scatter plot representing the mRNA enrichment in SGs in HeLa after si-neg or si-G3BP1 treatment and add-back of different RFP-G3BP1 constructs and GFP-Caprin1 (or GFP alone as control) and treatment with sodium arsenite. Each dot corresponds to the average mRNA enrichment in all SGs of all cells of a well expressing the RFP-G3BP1 and GFP-Caprin1 (or GFP alone) constructs. Data were obtained from four independent samples. The mean value is in red. Significances were obtained using t test; ∗p < 0.05; ∗∗∗p < 0.005. Same as (D) but the plot represents the G3BP1 (full length or truncations) enrichment in SGs after si-Neg or si-G3BP1 treatment. ∗∗∗p < 0.005; ns: Not significant. Scatter plot representing the mixing between G3BP1 full length and different G3BP1 truncations (including G3BP1 full length itself as a positive control) or YBX1 as a negative control. All G3BP1 constructs and YBX1 were brought on MTs. Each data point represents the determination coefficient R 2 obtained from all compartments detected on MT of all cells of a well. The plot shows the data from five to eight independent experiments. Red lines show mean values (in red also). Significances between mixing scores were obtained using t test; ∗∗∗p < 0.005. Same as ( F ) but the mixing score is obtained between Caprin1 (or YBX1 as negative control) and different G3BP1 truncations. Representative images of mixing between Caprin1 (or YBX1) and G3BP1 (FL and truncation) brought on MT.

Article Snippet: Plasmids encoding G3BP1, G3BP1-ΔC, G3BP1-ΔN and G3BP1-ΔIDR2 fused with RFP-tag on N-terminus were constructed by amplification corresponding cDNAs with primers containing appropriate digestion sites and inserted into the mRFP-C1 vector (Addgene).

Techniques: Over Expression, Expressing, Construct, Fluorescence, Control, Positive Control, Negative Control