c terminal v5 tag  (Addgene inc)

Journal: Molecular cell

Article Title: Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation

doi: 10.1016/j.molcel.2024.08.010

Figure Lengend Snippet: key resources table

Article Snippet: For eCLIP and Cut&Run experiments, ORFs were recombined into the R1 destination vector with a C-terminal V5 epitope tag expressed under an EF1-alpha promoter (Thermo Fisher #V602020).

Techniques: Virus, Recombinant, Transfection, Mutagenesis, Luciferase, Clone Assay, Mass Spectrometry, Microscopy, shRNA, Plasmid Preparation, Software

Journal: Nature Cell Biology

Article Title: VAMP2 regulates phase separation of α-synuclein

doi: 10.1038/s41556-024-01451-6

Figure Lengend Snippet: a , Co-expression of VAMP2 and wild-type αSYN–YFP in HeLa cells showing condensate formation. Cells co-expressing VAMP2 and αSYN(A30P)–YFP lack condensate formation. b , Quantification of cells forming condensates. Data are derived from Incucyte screening, with 16 images per well, three wells per biological repeat and four biological repeats. n indicates biological repeats. Data are mean ± s.d. One-way ANOVA with Dunnett’s multiple comparison test. c , Co-expression of αSYN–YFP, VAMP2 and mScarlet synaptotagmin showing partial co-localization of mScarlet synaptotagmin with αSYN–YFP condensates. d , Quantification of Pearson correlation coefficient for αSYN–YFP and mScarlet synaptotagmin co-localization. Three biological repeats were conducted; n indicates biological repeats. Data are mean ± s.d. Unpaired two-tailed t -test. e , Zoomed-in areas highlighting co-localization of αSYN condensates with co-expressed mScarlet synaptotagmin and mScarlet synaptotagmin outside αSYN condensates. Fluorescence intensity distribution for αSYN–YFP (yellow) and mScarlet synaptotagmin (magenta). f , Quantification of mScarlet synaptotagmin intensity outside and within αSYN condensates. n = 10 cells, pooled from four biological repeats. Data are mean ± s.d. Unpaired two-tailed t -test. g , HeLa cells with ectopic expression of αSYN–YFP, VAMP2 and 4xMTS-mScarlet, electron microscopy image overlaid with fluorescence microscopy, showing assemblies of vesicles colocalizing with αSYN–YFP condensates. Also see Extended Data Fig. for individual images. h , Electron microscopy images for individual vesicle clusters in Fig. 7g. Scale bar, 1 µm. i , Histogram showing size distribution of vesicles contained within αSYN condensates. Data are mean ± s.d. n = 14 vesicle clusters pooled from two cells from two biological repeats. j , Co-expression of αSYN–YFP, VAMP2 and complexin-1/2 mScarlet demonstrating enrichment of complexins within αSYN–YFP condensates. k , Quantification of complexin in αSYN condensates versus cytosolic complexin levels. Complexin-2 levels were significantly higher than complexin-1. n = 21 and 23 cells for complexin-1 and complexin-2, respectively, pooled from three biological repeats. Data are mean ± s.d. Unpaired two-tailed t -test.

Article Snippet: SYT1 , encoding synaptotagmin-1, was cloned from SH-SY5Y cDNA into the pCDNA3.1 vector with an N-terminal mScarlet tag, CPLX1 and CLPX2 , encoding complexin-1 and complexin-2 were cloned from SH-SY5Y cDNA into the pCDNA3.1 vector with a C-terminal mScarlet tag (Addgene 16015 and Addgene 85045).

Techniques: Expressing, Derivative Assay, Comparison, Two Tailed Test, Fluorescence, Electron Microscopy, Microscopy

Journal: The Journal of Cell Biology

Article Title: Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: Supplemental data on activity of RNF183 , FAM86B2, and SUGP1 crRNAs. (A) Indel distribution (from TIDE analysis) in sequences of target regions in unmodified U-2 OS cells transfected with VBC-derived ( V ) RNF183 crRNAs. (B) Incomplete targeting with the crRNF183 pool may be associated with an overlapping target region and/or spanning of a common polymorphism (rs3750534) within a target sequence for crRNF183 ( V ) #3 and crRNF183 ( V ) #4, which have particularly low activity. (C) Indel distribution (from TIDE analysis) in sequences of target regions in unmodified U-2 OS cells transfected with the VBC-derived ( V ) crFAM86B2 crRNA pool. Insufficient targeting of the crFAM86B2 pool may be due to all four crRNAs targeting overlapping regions, and therefore competing with each other. (D) Phenotyping of individual and pooled SUGP1 crRNAs. Heatmap displaying quantification of SUGP1 protein signal and localization ratio (perinuclear versus peripheral) of GFP-BICD2N-FRB and PTS-RFP-FKBP spots in U-2 OS PEX cells with the indicated treatments. “( D )” and “( V )” indicate source of crRNA design ( D , Discovery; V , VBC score). Data points represent mean per cell values aggregated at well levels (minimum of 100 cells analyzed per condition; four wells per condition). Color scale of individual features was adjusted based on minimum and maximum raw values. The guides selected for RNA-seq are labeled in gold text. (E–G) Quality control for samples submitted for RNA-seq. (E) Violin plots of intensity of SUGP1 protein signal at the single cell level (median, bold line; first/third quartile, dashed lines; minimum of 100 cells analyzed per condition). (F and G) Indel distribution (from TIDE analysis) in sequences of target regions in unmodified U-2 OS cells transfected with crSUGP1 #1 (F) or crXCR1 #1 (G). In A, C, F, and G, bar graphs display mean ± SD, and the efficiency scores are out of 100. Corresponding sequences from cells transfected with NTC crRNAs #1–4 (A and C) and NTC crRNA #1 (F and G) were used as a reference.

Article Snippet: The cDNA sequence for human SUGP1 (based on RefSeq: NM_172231) fused with a C-terminal V5 epitope tag was synthesized and cloned into the KpnI and XbaI restriction sites in pcDNA3.1(+) by Azenta Biosciences.

Techniques: Activity Assay, Transfection, Derivative Assay, Sequencing, RNA Sequencing, Labeling, Control

Journal: The Journal of Cell Biology

Article Title: Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: SUGP1 sustains functional levels of LIS1 mRNA and protein. (A) Schematic of SUGP1 domain structure. NLS, nuclear localization signal. (B) Representative images of SUGP1 intensity and GFP-BIC2N-FRB and PTS-RFP-FKBP localization in U-2 OS PEX cells treated with crSUGP1 #1. Scale bar, 25 µm. (C) Scatter plot of mRNA abundance for crSUGP1 #1-edited versus NTC-treated U-2 OS cells (mean log 2 normalized values from three independently performed experiments). mRNAs meeting the threshold for inclusion (minimum absolute log 2 fold change ≥0.5; FDR ≤ 0.05) are labeled in blue, except SUGP1 , DYNC1I2 , and LIS1 , which are labeled in yellow. The inset table shows non-logarithmic values for SUGP1 , DYNC1I2 , and LIS1 . See for full results. (D) Quantification of endogenous DYNC1I2 and LIS1 protein signal (determined by immunofluorescence) in unmodified U-2 OS cells treated with NTC or crSUGP1 #1 or #2 and transfected with a control (iRFP670) or crRNA-resistant SUGP1-V5 expression plasmid. (E) Representative images and quantification (perinuclear versus peripheral localization ratio) of GFP-BICD2N-FRB and PTS-RFP-FKBP localization in U-2 OS PEX cells treated with NTC or crSUGP1 #1 or #2 and transfected with a control (iRFP670), crRNA-resistant SUGP1-V5, or LIS1-FLAG expression plasmid. Scale bar, 25 µm. In D and E, cells were transfected with crRNA 96 h before fixation, and with expression plasmid 48 h after crRNA transfection. Data points represent mean per cell intensity values (D) or mean per cell localization ratio values (E) aggregated at well level from four independent experiments (minimum of 100 transfected cells analyzed per well; four wells analyzed per condition). Error bars signify SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA with Tukey’s multiple comparison; colors of asterisks indicate comparison group).

Article Snippet: The cDNA sequence for human SUGP1 (based on RefSeq: NM_172231) fused with a C-terminal V5 epitope tag was synthesized and cloned into the KpnI and XbaI restriction sites in pcDNA3.1(+) by Azenta Biosciences.

Techniques: Functional Assay, Labeling, Immunofluorescence, Transfection, Control, Expressing, Plasmid Preparation, Comparison

Journal: The Journal of Cell Biology

Article Title: Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: Supplemental data for differential expression and splicing analysis. (A) Scatter plot of mRNA abundance for (left panel) XCR1 -edited versus SUGP1 -edited U-2 OS cells and (right panel) NTC versus XCR1 -edited U-2 OS cells (mean log 2 normalized values from three independent experiments). mRNAs meeting threshold for inclusion (minimum absolute log 2 normalized fold change ≥0.5 and FDR ≤ 0.05) are labeled in blue, except (left panel) SUGP1 , DYNC1I2 , and LIS1 , and (right panel) XIRP1 (the only differentially expressed gene in the NTC versus crXCR1 comparison), which are labeled in yellow. Inset tables show non-logarithmic values for (left panel) SUGP1 , DYNC1I2 , and LIS1 and (right panel) XIRP1 mRNAs. See for full results. (B) Venn diagram showing overlap of differentially expressed genes in the NTC versus crSUGP1 and crXCR1 versus crSUGP1 comparisons. (C) Quantification of LIS1 and DYNC1I2 mRNA level, determined by TaqMan-based real-time qPCR, in SUGP1 -edited and XCR1 -edited U-2 OS and ARPE-19 cells. Data points represent the mean of three independent experiments (RQ = relative quantification based on NTC). Error bars signify SD. *P < 0.05, **P < 0.01 (one-way ANOVA with Dunnett’s multiple comparison against NTC). (D and E) Venn diagrams showing the overlap of genes that undergo differential splicing (D) and differential splicing events (E) in the datasets, as determined with rMATs (note that some genes have >1 differential splicing event). The threshold for classifying an event as differential was: absolute IncLevelDifference ≥0.2, total read count (inclusion count + skipping count) ≥10, and FDR ≤ 0.05. See for full results. (F) Classes of alternative splicing events common to both comparisons (i.e., NTC versus crSUGP1 or crXCR1 versus crSUGP1 ), as identified by rMATS. Blue and green represent events that were enriched in control and crSUGP1 samples, respectively. rMATS reports 5 splicing categories: (i) alternative 3′ splice sites (A3SS); (ii) alternative 5′ splice sites (A5SS); (iii) mutually exclusive exons (MXE); (iv) retained introns (RI), and (v) skipped exons (SE). The A3SS and A5SS events involve the splicing together of two exons separated by a single intron. For A3SS events, alternative splicing causes a downstream exon to extend partially into neighboring intronic sequence. A5SS is defined by an alternative splicing event causing an upstream exon to extend partially into the adjoining intron. MXEs describe the splicing of adjacent exons (separated by a single intron) in which one exon is retained but the other is excluded, or vice versa. The graph reports MXE events in which the upstream exon was selected. Events classified as RI are those in which an intron is not spliced out and hence is retained in the mature transcript. SE denotes splicing events in which an exon is skipped over and not included in the processed RNA molecule. (G) Venn diagrams showing overlap of differential 3′-end usage events in the datasets, as determined by LABRAT. LABRAT quantifies alternative polyadenylation sites and reports upstream or downstream shifts in the usage of those sites for each gene as compared to the control (see for full results). The threshold for classifying an event as differential was Δψ ≥0.05 and FDR ≤ 0.05.

Article Snippet: The cDNA sequence for human SUGP1 (based on RefSeq: NM_172231) fused with a C-terminal V5 epitope tag was synthesized and cloned into the KpnI and XbaI restriction sites in pcDNA3.1(+) by Azenta Biosciences.

Techniques: Quantitative Proteomics, Labeling, Comparison, Alternative Splicing, Control, Sequencing