hdr template phdr_g3bp1-mscarlet  (Addgene inc)

Journal: bioRxiv

Article Title: Ribosomal protein L5 (RPL5/uL18) I60V mutation is associated to increased translation and modulates drug sensitivity in T-cell acute lymphoblastic leukemia cells

doi: 10.1101/2025.07.16.665036

Figure Lengend Snippet:

Article Snippet: RNP complexes and 5 μg of single strand HDR donor template (ssODN, ) were mixed with 10 6 cells to a final volume of 100 μl in EP buffer (Optimem medium, Thermofisher) and electroporated by using Nepa21 electroporator system (Nepa Gene Co., Ltd) using the following settings for poring: pulse 175V, 5 ms length, 50 ms interval, and manufacturer’s recommended protocol for transfer pulse.

Techniques: Synthesized, Knock-In

Journal: bioRxiv

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

doi: 10.1101/2024.10.24.620053

Figure Lengend Snippet: A) Schematic depicting possible SG localization of the UBQLN2-RTL8-PEG10 complex given the known SG localization of UBQLN2 and PEG10. B) PLA assessing the interaction between endogenous UBQLN2 and PEG10 in HeLa cells induced to form SGs by sodium arsenite or heat shock. Nuclei and cell membranes are labeled with DAPI and WGA, respectively, and arrowheads in insets mark SGs stained with G3BP1. Quantification of PLA foci/μm within SGs and in the whole cell is shown below. Number of individual cells quantified per condition from 2 independent replicates is indicated. One-way ANOVA with Tukey’s multiple comparison test was performed. Scale bars: 10 µm and 5 µm for insets. Also, see . C) Biotinylated isoxazole (B-isox)-mediated precipitation of RNA granule proteins from three human cell lines. WCL = whole cell lysate before B-isox addition, S = supernatant, and P = pellet. G3BP1 and TIAR are positive controls, GAPDH is a negative control. Ponceau S was used to normalize loading. D) 3-color dSTORM of HeLa cells treated with sodium arsenite. Numbers identify the zoomed regions shown below. Arrowheads mark regions of overlap between PEG10 and G3BP1. Jaccard index between G3BP1 clusters and UBQLN2 or PEG10 is shown at top right. Dotted lines connect data points from an individual cell. An unpaired t-test was performed. N=15 cells across 3 independent replicates. Scale bars: 10 µm and 0.5 µm for zoomed regions.

Article Snippet: The guide RNA sequences were as follows: sgNon-targeting: 5’-GATACGTCGGTACCGGACCG-3’ sgRTL8: 5’-GTTCTCGTCCACGAACATGT-3’ sgPEG10 gag-pol: 5’-GATCATGGCTCGGACGAACA-3’ sgPEG10: 5’-GCTGATTGACCAGTACCACG-3’ sgUBQLN2: 5’-ACGCAGCCTAGCAATGCCGC-3’ sgG3BP1: 5’-TCCATGAAGATTCACTGCCG-3’ For CRISPR knock-in of a V5-APEX2-mScarlet-I tag into the endogenous G3BP1 locus, the generated Cas9-sgG3BP1 plasmid (template Addgene #118055) was co-transfected with the HDR template pHDR_G3BP1-mScarlet (Addgene #196196) at a 1:1 ratio into the various HeLa KO lines.

Techniques: Labeling, Staining, Comparison, Negative Control

Journal: bioRxiv

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

doi: 10.1101/2024.10.24.620053

Figure Lengend Snippet: A) After treatment with sodium arsenite, the indicated HeLa cell lines were immunostained for endogenous UBQLN2, PEG10, and the SG marker G3BP1. Arrowheads in insets mark SGs. UBQLN2 or PEG10 intensity within SGs was quantified and is shown to the right. Also, see Figures S4 and S5A. B) NTC and RTL8 KO HeLa cells expressing HA-RTL8C, HA-RTL8C ΔN, or control protein (HA-mFAP10), treated with sodium arsenite. UBQLN2 intensity within SGs is shown at bottom left. The Mander’s overlap coefficient (MOC) between HA and G3BP1 is shown at bottom right. C) U-2 OS G3BP1-GFP cells expressing V5-PEG10 gag, gag-pol, gag-pol ΔPPR, or control protein (V5-Nanoluciferase) treated with sodium arsenite. Immunoblot of lysates from HeLa and U-2 OS G3BP1-GFP cells is shown at bottom left. UBQLN2 intensity within SGs is shown at bottom right. Also, see . In (A-C), nuclei are labeled with DAPI. Scale bars: 10 µm and 5 µm for insets. In (B, C), filled arrowheads in insets indicates SGs with UBQLN2, unfilled arrows indicate SG without UBQLN2. In all panels, one-way ANOVA with Tukey’s multiple comparison test was performed. The number of individual cells quantified per line/plasmid from 3 independent replicates is indicated.

Article Snippet: The guide RNA sequences were as follows: sgNon-targeting: 5’-GATACGTCGGTACCGGACCG-3’ sgRTL8: 5’-GTTCTCGTCCACGAACATGT-3’ sgPEG10 gag-pol: 5’-GATCATGGCTCGGACGAACA-3’ sgPEG10: 5’-GCTGATTGACCAGTACCACG-3’ sgUBQLN2: 5’-ACGCAGCCTAGCAATGCCGC-3’ sgG3BP1: 5’-TCCATGAAGATTCACTGCCG-3’ For CRISPR knock-in of a V5-APEX2-mScarlet-I tag into the endogenous G3BP1 locus, the generated Cas9-sgG3BP1 plasmid (template Addgene #118055) was co-transfected with the HDR template pHDR_G3BP1-mScarlet (Addgene #196196) at a 1:1 ratio into the various HeLa KO lines.

Techniques: Marker, Expressing, Control, Western Blot, Labeling, Comparison, Plasmid Preparation

Journal: bioRxiv

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

doi: 10.1101/2024.10.24.620053

Figure Lengend Snippet: A) WT HeLa cells treated with non-targeting control siRNA or siRNA against RTL8, PEG10 or UBQLN2, then treated with sodium arsenite. B) NTC, PEG10 KO and PEG10 gag-pol KO HeLa cells treated with sodium arsenite are shown on the right. Immunoblot of lysates from respective cell lines probed for the indicated proteins are shown on the left. GAPDH = loading control. In (A & B), cells are stained for endogenous UBQLN2, PEG10, and G3BP1. Nuclei are labeled with DAPI and arrowheads in insets mark SGs. Scale bars: 10 µm and 5 µm for insets.

Article Snippet: The guide RNA sequences were as follows: sgNon-targeting: 5’-GATACGTCGGTACCGGACCG-3’ sgRTL8: 5’-GTTCTCGTCCACGAACATGT-3’ sgPEG10 gag-pol: 5’-GATCATGGCTCGGACGAACA-3’ sgPEG10: 5’-GCTGATTGACCAGTACCACG-3’ sgUBQLN2: 5’-ACGCAGCCTAGCAATGCCGC-3’ sgG3BP1: 5’-TCCATGAAGATTCACTGCCG-3’ For CRISPR knock-in of a V5-APEX2-mScarlet-I tag into the endogenous G3BP1 locus, the generated Cas9-sgG3BP1 plasmid (template Addgene #118055) was co-transfected with the HDR template pHDR_G3BP1-mScarlet (Addgene #196196) at a 1:1 ratio into the various HeLa KO lines.

Techniques: Control, Western Blot, Staining, Labeling

Journal: bioRxiv

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

doi: 10.1101/2024.10.24.620053

Figure Lengend Snippet: A) Schematic of live-cell imaging experiments in HeLa cell lines expressing endogenous G3BP1-mScarlet-I. Nuclei are labeled with Hoechst 33342. Live-cell imaging showing timepoints during arsenite stress ( B ) and recovery after wash out ( C ). Shown below are SG counts per cell over time. Data represent means ± SEM. SGs/cell are normalized to the maximum number of SGs observed under each condition, fitted to a non-linear regression model and analyzed with one-way ANOVA with Tukey’s multiple comparison test. N=24 fields across 3 independent replicates. Scale bar: 10 µm. Also, see and Movies S1 and S2.

Article Snippet: The guide RNA sequences were as follows: sgNon-targeting: 5’-GATACGTCGGTACCGGACCG-3’ sgRTL8: 5’-GTTCTCGTCCACGAACATGT-3’ sgPEG10 gag-pol: 5’-GATCATGGCTCGGACGAACA-3’ sgPEG10: 5’-GCTGATTGACCAGTACCACG-3’ sgUBQLN2: 5’-ACGCAGCCTAGCAATGCCGC-3’ sgG3BP1: 5’-TCCATGAAGATTCACTGCCG-3’ For CRISPR knock-in of a V5-APEX2-mScarlet-I tag into the endogenous G3BP1 locus, the generated Cas9-sgG3BP1 plasmid (template Addgene #118055) was co-transfected with the HDR template pHDR_G3BP1-mScarlet (Addgene #196196) at a 1:1 ratio into the various HeLa KO lines.

Techniques: Live Cell Imaging, Expressing, Labeling, Comparison

Journal: bioRxiv

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

doi: 10.1101/2024.10.24.620053

Figure Lengend Snippet: A) Immunoblot of lysates from the indicated cell lines, where endogenous G3BP1 has been CRISPR-tagged with V5-APEX2-mScarlet-I. Lysate from untagged NTC HeLa cells is also included for comparison. GAPDH = loading control. B) Area and intensity of SGs from the indicated cell lines in after 20 minutes of arsenite treatment as shown in . A one-way ANOVA with Tukey’s multiple comparison test was performed. N= 24 fields across 3 independent replicates.

Article Snippet: The guide RNA sequences were as follows: sgNon-targeting: 5’-GATACGTCGGTACCGGACCG-3’ sgRTL8: 5’-GTTCTCGTCCACGAACATGT-3’ sgPEG10 gag-pol: 5’-GATCATGGCTCGGACGAACA-3’ sgPEG10: 5’-GCTGATTGACCAGTACCACG-3’ sgUBQLN2: 5’-ACGCAGCCTAGCAATGCCGC-3’ sgG3BP1: 5’-TCCATGAAGATTCACTGCCG-3’ For CRISPR knock-in of a V5-APEX2-mScarlet-I tag into the endogenous G3BP1 locus, the generated Cas9-sgG3BP1 plasmid (template Addgene #118055) was co-transfected with the HDR template pHDR_G3BP1-mScarlet (Addgene #196196) at a 1:1 ratio into the various HeLa KO lines.

Techniques: Western Blot, CRISPR, Comparison, Control

Journal: bioRxiv

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

doi: 10.1101/2024.10.24.620053

Figure Lengend Snippet: A) Schematic of SG pulldown experiments from HeLa cell lines. Also, see . B) Venn diagrams showing significantly decreased or enriched SG proteins in the indicated HeLa cell lines. SG protein tiers are annotated based on the RNA granule database. Also, see and Data S2. C) Heatmap of known PEG10 interactors detected in SG IP/MS experiments. Bolded proteins are also SG proteins. Colors correspond to fold enrichment in respective KO line versus NTC. Black box identifies TCAF1 and ATXN10, and pink box identifies TSG101, an EV marker. Also, see and . D) After treatment with sodium arsenite, the indicated cell lines were stained for endogenous ATXN10 and G3BP1. Nuclei are labeled with DAPI and arrowheads in insets mark SGs. Scale bars: 10 µm and 5 µm for insets. Also, see .

Article Snippet: The guide RNA sequences were as follows: sgNon-targeting: 5’-GATACGTCGGTACCGGACCG-3’ sgRTL8: 5’-GTTCTCGTCCACGAACATGT-3’ sgPEG10 gag-pol: 5’-GATCATGGCTCGGACGAACA-3’ sgPEG10: 5’-GCTGATTGACCAGTACCACG-3’ sgUBQLN2: 5’-ACGCAGCCTAGCAATGCCGC-3’ sgG3BP1: 5’-TCCATGAAGATTCACTGCCG-3’ For CRISPR knock-in of a V5-APEX2-mScarlet-I tag into the endogenous G3BP1 locus, the generated Cas9-sgG3BP1 plasmid (template Addgene #118055) was co-transfected with the HDR template pHDR_G3BP1-mScarlet (Addgene #196196) at a 1:1 ratio into the various HeLa KO lines.

Techniques: Marker, Staining, Labeling

Journal: bioRxiv

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

doi: 10.1101/2024.10.24.620053

Figure Lengend Snippet: A) Immunoblot of lysates from untreated and arsenite-treated HeLa cells subjected to SG pulldown with ⍺-G3BP1 Ig. ATXN2 and TIAR are positive controls, GAPDH is a negative control. B) Volcano plot of proteins significantly increased (red) or decreased (blue) in the indicated KO HeLa line compared to NTC cells. N = 4 biological replicates. The cutoff was set at p <0.05 and absolute fold change of 2. Labels and symbols are based on annotations of SG protein tiers on the RNA granule database. Boxes identify TCAF1 and ATXN10. C) Heat map of commonly used EV markers detected in our SG IP/MS experiments. Bolded hits are also SG proteins. Colors correspond to fold enrichment in respective KO line versus NTC. Black box identifies TCAF1 and ATXN10 and pink box identifies the PEG10 interactor, TSG101. D) At left, immunoblots of lysates from the indicated cell lines probed for TCAF1 and ATXN10. At right, relative TCAF1 and ATXN10 levels in the indicated lines, normalized to levels in NTC cells. Data represent means ± SD (N=3), analyzed with one-way ANOVA with Tukey’s multiple comparison test. GAPDH = loading control, * denotes non-specific band. E) Sodium arsenite-treated NTC, PEG10 KO and PEG10 gag-pol KO HeLa cells were stained for endogenous ATXN10 and G3BP1. Nuclei are labeled with DAPI and arrowheads in insets mark SGs. Scale bars: 10 µm and 5 µm for insets.

Article Snippet: The guide RNA sequences were as follows: sgNon-targeting: 5’-GATACGTCGGTACCGGACCG-3’ sgRTL8: 5’-GTTCTCGTCCACGAACATGT-3’ sgPEG10 gag-pol: 5’-GATCATGGCTCGGACGAACA-3’ sgPEG10: 5’-GCTGATTGACCAGTACCACG-3’ sgUBQLN2: 5’-ACGCAGCCTAGCAATGCCGC-3’ sgG3BP1: 5’-TCCATGAAGATTCACTGCCG-3’ For CRISPR knock-in of a V5-APEX2-mScarlet-I tag into the endogenous G3BP1 locus, the generated Cas9-sgG3BP1 plasmid (template Addgene #118055) was co-transfected with the HDR template pHDR_G3BP1-mScarlet (Addgene #196196) at a 1:1 ratio into the various HeLa KO lines.

Techniques: Western Blot, Negative Control, Comparison, Control, Staining, Labeling

Journal: bioRxiv

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties

doi: 10.1101/2024.10.24.620053

Figure Lengend Snippet: A) Schematic of the cryo-CLEM workflow for imaging PEG10 gag-pol-Dendra2-labeled VLPs in cells (i.e. in situ) or isolated from cell culture media (VLP suspension). B) Representative maximum intensity projection (MIP) of vitrified VLPs from PEG10 KO HeLa cells transfected with PEG10 gag-pol-Dendra2 overlaid on the reflection channel image. The inset depicts a low-magnification TEM image targeting the Dendra2 fluorescence signal. Also, see Figures S8A-C. Scale bars: 20 µm and 0.5 µm for overlay and inset images, respectively. C) Slice through a tomogram of the region highlighted in B showing two representative PEG10 gag-pol-Dendra2 VLPs. Scale bar: 50 nm. D) Representative overlay of MIP of cryo-confocal Z-stack with the image from the reflection channel (gray) of arsenite-treated vitrified PEG10 KO HeLa cells expressing endogenous G3BP1-mScarlet-I, transfected with PEG10 gag-pol-Dendra2. Fiducial fluorospheres (blue) were added for correlation. Scale bar: 20 µm. Also, see . E) Cryo-focused ion beam view post-milling with transformed cryo-confocal MIP matching the 15° milling angle. Scale bar: 10 µm. F) Overlay of medium magnification montage from cryo-TEM (6500x) of a lamella with cryo-confocal MIP. The solid black rectangle indicates where the tilt series was subsequently acquired. Scale bar: 2 µm. G) A slice through the tomogram of the region highlighted in F was collected at 42,000x, revealing well-preserved cellular interiors. Four (1-4) PEG10 gag-pol-Dendra2 VLPs in the field of view are highlighted in white dashed rectangles. Enlarged views of the four VLPs are also shown to highlight the variability in structure and morphology, such as additional protein coat (orange arrowheads, rectangle 3). Scale bar: 200 nm. Also, see . H) Segmentation of the tomogram volume shown in G. Stress granules (pink), PEG10 gag-pol-Dendra2 VLP (red), ribosomes (blue), intermediate filaments (yellow), and microtubules (green) are highlighted. Also, see Movie S3.

Article Snippet: The guide RNA sequences were as follows: sgNon-targeting: 5’-GATACGTCGGTACCGGACCG-3’ sgRTL8: 5’-GTTCTCGTCCACGAACATGT-3’ sgPEG10 gag-pol: 5’-GATCATGGCTCGGACGAACA-3’ sgPEG10: 5’-GCTGATTGACCAGTACCACG-3’ sgUBQLN2: 5’-ACGCAGCCTAGCAATGCCGC-3’ sgG3BP1: 5’-TCCATGAAGATTCACTGCCG-3’ For CRISPR knock-in of a V5-APEX2-mScarlet-I tag into the endogenous G3BP1 locus, the generated Cas9-sgG3BP1 plasmid (template Addgene #118055) was co-transfected with the HDR template pHDR_G3BP1-mScarlet (Addgene #196196) at a 1:1 ratio into the various HeLa KO lines.

Techniques: Imaging, Labeling, In Situ, Isolation, Cell Culture, Suspension, Transfection, Fluorescence, Expressing, Transformation Assay