human cell line hek293  (ATCC)


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    ATCC human cell line hek293
    Human Cell Line Hek293, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hek293 cell lines  (ATCC)


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    ATCC hek293 cell lines
    A representative IV curves induced by the −100 to +100 mV voltage ramp recorded in <t>HEK</t> cells transfected with the vEF1ap-5’UTR-TRPV6wt_CMVp-GFP vector. Curves show whole-cell TRPV6 currents in either base HBSS/basal medium (black), DVF medium alone (red), or DVF medium containing either 12 µg/ml of mAb82 anti-TRPV6 antibody (pink) or 24 µg/ml (yellow). B representative trace of the whole-cell currents during the application of the DVF solution as well as different doses of mAb82 antibody, as indicated by arrows. C bar plots summarizing average whole-cell currents under conditions indicated above for mAb82, ( n = 15 for 1.2 µg/ml; n = 20 for 2.4 µg/ml; n = 12 for 6 µg/ml; n = 11 for 12 µg/ml; and n = 11 for 24 µg/ml). D SOCE in the LNCaP cells pretreated 5 min with either mouse monoclonal mAbAU1 as a control antibody or a mouse anti-human TRPV6 mAb82 antibody, both at 2.4 µg/ml. E corresponding quantitative representation of the ER content (calculated as a maximum amplitude), n = 3, * p < 0.05; the SOCE affected by antibody-induced treatments shown in ( D ); n = 3, *** p < 0.001; and the slope of SOCE calculated as a ratio delta/sec for each condition; n = 3, * p < 0.05.
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    1) Product Images from "Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression"

    Article Title: Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-024-06809-0

    A representative IV curves induced by the −100 to +100 mV voltage ramp recorded in HEK cells transfected with the vEF1ap-5’UTR-TRPV6wt_CMVp-GFP vector. Curves show whole-cell TRPV6 currents in either base HBSS/basal medium (black), DVF medium alone (red), or DVF medium containing either 12 µg/ml of mAb82 anti-TRPV6 antibody (pink) or 24 µg/ml (yellow). B representative trace of the whole-cell currents during the application of the DVF solution as well as different doses of mAb82 antibody, as indicated by arrows. C bar plots summarizing average whole-cell currents under conditions indicated above for mAb82, ( n = 15 for 1.2 µg/ml; n = 20 for 2.4 µg/ml; n = 12 for 6 µg/ml; n = 11 for 12 µg/ml; and n = 11 for 24 µg/ml). D SOCE in the LNCaP cells pretreated 5 min with either mouse monoclonal mAbAU1 as a control antibody or a mouse anti-human TRPV6 mAb82 antibody, both at 2.4 µg/ml. E corresponding quantitative representation of the ER content (calculated as a maximum amplitude), n = 3, * p < 0.05; the SOCE affected by antibody-induced treatments shown in ( D ); n = 3, *** p < 0.001; and the slope of SOCE calculated as a ratio delta/sec for each condition; n = 3, * p < 0.05.
    Figure Legend Snippet: A representative IV curves induced by the −100 to +100 mV voltage ramp recorded in HEK cells transfected with the vEF1ap-5’UTR-TRPV6wt_CMVp-GFP vector. Curves show whole-cell TRPV6 currents in either base HBSS/basal medium (black), DVF medium alone (red), or DVF medium containing either 12 µg/ml of mAb82 anti-TRPV6 antibody (pink) or 24 µg/ml (yellow). B representative trace of the whole-cell currents during the application of the DVF solution as well as different doses of mAb82 antibody, as indicated by arrows. C bar plots summarizing average whole-cell currents under conditions indicated above for mAb82, ( n = 15 for 1.2 µg/ml; n = 20 for 2.4 µg/ml; n = 12 for 6 µg/ml; n = 11 for 12 µg/ml; and n = 11 for 24 µg/ml). D SOCE in the LNCaP cells pretreated 5 min with either mouse monoclonal mAbAU1 as a control antibody or a mouse anti-human TRPV6 mAb82 antibody, both at 2.4 µg/ml. E corresponding quantitative representation of the ER content (calculated as a maximum amplitude), n = 3, * p < 0.05; the SOCE affected by antibody-induced treatments shown in ( D ); n = 3, *** p < 0.001; and the slope of SOCE calculated as a ratio delta/sec for each condition; n = 3, * p < 0.05.

    Techniques Used: Transfection, Plasmid Preparation

    hek293 cells  (ATCC)


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    ATCC hek293 cells
    Enzyme-mediated C-terminal cleavage exposes the CAD. (A) A schematic representation of the full length Panx1 harboring a TEV protease cleavage site instead of the caspase recognition site (Panx1-T). (B) and (C) Exemplar whole cell currents (B) and peak current density at +110 mV (C) of Panx1-T co-expressed in <t>HEK293</t> cells with TEV protease. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=4. Asterisks indicate significance of p<0.05 determined by student T-test. (D) Inside-out patch clamp recordings with or without recombinant TEV protease (0.1mg/mL). Voltage ramps from 130 mV to +80 mV over 0.5 s were applied every 6 s. Representative peak currents at +80 mV are plotted for Panx1-T (blue) and Panx1-T L367A/L370A (gray). CBX (100 μM) were applied at time points indicated by orange bars. (E) TEV specific currents defined as the peak current differences between the indicated time points a and b . N=4. Asterisk indicates a p<0.05 obtained from an unpaired student T-test.
    Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The C-terminal activating domain promotes Panx1 channel opening"

    Article Title: The C-terminal activating domain promotes Panx1 channel opening

    Journal: bioRxiv

    doi: 10.1101/2024.06.13.598903

    Enzyme-mediated C-terminal cleavage exposes the CAD. (A) A schematic representation of the full length Panx1 harboring a TEV protease cleavage site instead of the caspase recognition site (Panx1-T). (B) and (C) Exemplar whole cell currents (B) and peak current density at +110 mV (C) of Panx1-T co-expressed in HEK293 cells with TEV protease. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=4. Asterisks indicate significance of p<0.05 determined by student T-test. (D) Inside-out patch clamp recordings with or without recombinant TEV protease (0.1mg/mL). Voltage ramps from 130 mV to +80 mV over 0.5 s were applied every 6 s. Representative peak currents at +80 mV are plotted for Panx1-T (blue) and Panx1-T L367A/L370A (gray). CBX (100 μM) were applied at time points indicated by orange bars. (E) TEV specific currents defined as the peak current differences between the indicated time points a and b . N=4. Asterisk indicates a p<0.05 obtained from an unpaired student T-test.
    Figure Legend Snippet: Enzyme-mediated C-terminal cleavage exposes the CAD. (A) A schematic representation of the full length Panx1 harboring a TEV protease cleavage site instead of the caspase recognition site (Panx1-T). (B) and (C) Exemplar whole cell currents (B) and peak current density at +110 mV (C) of Panx1-T co-expressed in HEK293 cells with TEV protease. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=4. Asterisks indicate significance of p<0.05 determined by student T-test. (D) Inside-out patch clamp recordings with or without recombinant TEV protease (0.1mg/mL). Voltage ramps from 130 mV to +80 mV over 0.5 s were applied every 6 s. Representative peak currents at +80 mV are plotted for Panx1-T (blue) and Panx1-T L367A/L370A (gray). CBX (100 μM) were applied at time points indicated by orange bars. (E) TEV specific currents defined as the peak current differences between the indicated time points a and b . N=4. Asterisk indicates a p<0.05 obtained from an unpaired student T-test.

    Techniques Used: Patch Clamp, Recombinant

    CAD can facilitate Panx1 activation from non-native positions. Schematic representations ((A), (C), (F), and (H)), representative whole cell recordings ((B), (D), (G), (I)), and peak current density at +110 mV ((E) and (J)) are shown for each tested construct. HEK293 cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=5-24 and error bars indicate SEM. Asterisks indicate significance of p<0.05 determined by one-way ANOVA followed by Dunnett’s test comparing the Panx1Δ356 (E) or Panx1 wild type (J) to each construct.
    Figure Legend Snippet: CAD can facilitate Panx1 activation from non-native positions. Schematic representations ((A), (C), (F), and (H)), representative whole cell recordings ((B), (D), (G), (I)), and peak current density at +110 mV ((E) and (J)) are shown for each tested construct. HEK293 cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=5-24 and error bars indicate SEM. Asterisks indicate significance of p<0.05 determined by one-way ANOVA followed by Dunnett’s test comparing the Panx1Δ356 (E) or Panx1 wild type (J) to each construct.

    Techniques Used: Activation Assay, Construct

    Docked anions in the permeation pathway contribute to the channel closure. (A) Electrostatic free energy calculation of frPanx1-ΔC (red) in the presence of 1-4 ATP molecules in the deep energy well. (B) A docked ATP molecule on the NTD-flipped down human Panx1 (PDB: 7F8N). (C) Close-up view of the putative ATP binding pocket. Positive residues surrounding the docked ATP are indicated. (D) Peak current density at +110 mV of the wild type and alanine substitutions expressed in HEK293 cells. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s with 20 mV increments. N=4-15 and error bars indicate SEM. Asterisks indicate significance of p<0.05 determined by one-way ANOVA followed by Dunnett’s test comparing WT to each construct.
    Figure Legend Snippet: Docked anions in the permeation pathway contribute to the channel closure. (A) Electrostatic free energy calculation of frPanx1-ΔC (red) in the presence of 1-4 ATP molecules in the deep energy well. (B) A docked ATP molecule on the NTD-flipped down human Panx1 (PDB: 7F8N). (C) Close-up view of the putative ATP binding pocket. Positive residues surrounding the docked ATP are indicated. (D) Peak current density at +110 mV of the wild type and alanine substitutions expressed in HEK293 cells. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s with 20 mV increments. N=4-15 and error bars indicate SEM. Asterisks indicate significance of p<0.05 determined by one-way ANOVA followed by Dunnett’s test comparing WT to each construct.

    Techniques Used: Binding Assay, Construct

    hek293 cells  (ATCC)


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    ATCC hek293 cells
    A . High confidence cross-links quantified in each biological replicate. B . Distribution of log 2 ratios of quantified cross-links in each biological replicate. C . Correlation between biological replicates of MCF-7 and HeLa comparison, Pearson’s R 2 = 0.88. D . Correlation between biological replicates of <t>HEK293</t> and HeLa comparison, Pearson’s R 2 = 0.91. Volcano plots with log 2 ratio of cross-links quantified with 95% confidence ≤ 0.5 in both biological replicates in MCF-7 to HeLa comparison ( E ) or with 95% confidence ≤ 0.5 in at least 3 distinct biological replicates in HEK293 and HeLa comparison ( F ) based on all contributing ions. Bonferroni corrected p -value of 0.05 and |log 2 FC| > 0.5 are used to indicate significance. G. Correlation between log 2 ratios for intra-protein cross-links and respective log 2 ratios for proteins based on whole proteome quantitation for HEK293 and HeLa comparison with R 2 = 0.52 and MCF-7 and HeLa comparison with R 2 = 0.68.
    Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines"

    Article Title: Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines

    Journal: bioRxiv

    doi: 10.1101/2024.06.12.598691

    A . High confidence cross-links quantified in each biological replicate. B . Distribution of log 2 ratios of quantified cross-links in each biological replicate. C . Correlation between biological replicates of MCF-7 and HeLa comparison, Pearson’s R 2 = 0.88. D . Correlation between biological replicates of HEK293 and HeLa comparison, Pearson’s R 2 = 0.91. Volcano plots with log 2 ratio of cross-links quantified with 95% confidence ≤ 0.5 in both biological replicates in MCF-7 to HeLa comparison ( E ) or with 95% confidence ≤ 0.5 in at least 3 distinct biological replicates in HEK293 and HeLa comparison ( F ) based on all contributing ions. Bonferroni corrected p -value of 0.05 and |log 2 FC| > 0.5 are used to indicate significance. G. Correlation between log 2 ratios for intra-protein cross-links and respective log 2 ratios for proteins based on whole proteome quantitation for HEK293 and HeLa comparison with R 2 = 0.52 and MCF-7 and HeLa comparison with R 2 = 0.68.
    Figure Legend Snippet: A . High confidence cross-links quantified in each biological replicate. B . Distribution of log 2 ratios of quantified cross-links in each biological replicate. C . Correlation between biological replicates of MCF-7 and HeLa comparison, Pearson’s R 2 = 0.88. D . Correlation between biological replicates of HEK293 and HeLa comparison, Pearson’s R 2 = 0.91. Volcano plots with log 2 ratio of cross-links quantified with 95% confidence ≤ 0.5 in both biological replicates in MCF-7 to HeLa comparison ( E ) or with 95% confidence ≤ 0.5 in at least 3 distinct biological replicates in HEK293 and HeLa comparison ( F ) based on all contributing ions. Bonferroni corrected p -value of 0.05 and |log 2 FC| > 0.5 are used to indicate significance. G. Correlation between log 2 ratios for intra-protein cross-links and respective log 2 ratios for proteins based on whole proteome quantitation for HEK293 and HeLa comparison with R 2 = 0.52 and MCF-7 and HeLa comparison with R 2 = 0.68.

    Techniques Used: Comparison, Quantitation Assay

    A . Workflow for processing cross-linked cells. B . Correlation plot between MCF-7/HeLa and HEK293/HeLa replicates. C . Difference to the mean for quantitation of cross-links corresponding to same residue pairs for HEK293/HeLa (left) and MCF-7/HeLa (right) datasets. D . Heatmap of cross-links common between MCF-7/HeLa and HEK293/HeLa datasets. E . Distribution of log 2 intensities for DIA data in all three cell lines. F . PCA plot based of DIA data shows clustering of biological replicates according to cell line. G . Volcano plots of protein level fold changes and Benjamin-Hochberg corrected p -values with 0.05 cutoff for significance.
    Figure Legend Snippet: A . Workflow for processing cross-linked cells. B . Correlation plot between MCF-7/HeLa and HEK293/HeLa replicates. C . Difference to the mean for quantitation of cross-links corresponding to same residue pairs for HEK293/HeLa (left) and MCF-7/HeLa (right) datasets. D . Heatmap of cross-links common between MCF-7/HeLa and HEK293/HeLa datasets. E . Distribution of log 2 intensities for DIA data in all three cell lines. F . PCA plot based of DIA data shows clustering of biological replicates according to cell line. G . Volcano plots of protein level fold changes and Benjamin-Hochberg corrected p -values with 0.05 cutoff for significance.

    Techniques Used: Quantitation Assay, Residue

    A . Heatmap of log 2 ratios of keratin cross-link levels produced in live cells showing that all K2C8 and K1C18 intra- and inter-link levels in HEK293 and MCF-7 cells show opposing changes, relative to link levels in HeLa cells. B . Spectra of a cross-link between K119 on keratin 18 and K130 on keratin 8 in HEK293/HeLa and MCF-7/HeLa are displayed as mirror images. Peaks for reporter ions (orange), peptide A (blue), and peptide B (purple) are shown on the original spectra. C . Isotopic envelops with the apportionment from RH and stump SH for peptides A and B show the opposite direction of change in HEK293 cells and MCF-7 cells compared to HeLa. In HEK293 (cross-linked with SH), the cross-link level is decreased relative to HeLa (cross-linked with RH). In MCF-7 (cross-linked with SH), the same cross-link shows the opposite change relative to HeLa (cross-linked with RH). In all plots, darker shaded color indicates apportioned intensity from SH sample, and lighter shading indicates RH samples. D . Boxplot of log 2 intensities for keratin 18 and keratin 8 protein levels in all three cell lines with significance as determined by Student’s t -test with multiple testing correction (* = 0.05, ** = 0.01). Each point indicates a value for either K1C18 (black) or K2C8 (grey) in each biological replicate. E . Immunofluorescence imaging with anti-keratin 18 (green) and anti-keratin 8 (red) antibodies in all cell lines indicating protein level differences consistent with proteome measurements.
    Figure Legend Snippet: A . Heatmap of log 2 ratios of keratin cross-link levels produced in live cells showing that all K2C8 and K1C18 intra- and inter-link levels in HEK293 and MCF-7 cells show opposing changes, relative to link levels in HeLa cells. B . Spectra of a cross-link between K119 on keratin 18 and K130 on keratin 8 in HEK293/HeLa and MCF-7/HeLa are displayed as mirror images. Peaks for reporter ions (orange), peptide A (blue), and peptide B (purple) are shown on the original spectra. C . Isotopic envelops with the apportionment from RH and stump SH for peptides A and B show the opposite direction of change in HEK293 cells and MCF-7 cells compared to HeLa. In HEK293 (cross-linked with SH), the cross-link level is decreased relative to HeLa (cross-linked with RH). In MCF-7 (cross-linked with SH), the same cross-link shows the opposite change relative to HeLa (cross-linked with RH). In all plots, darker shaded color indicates apportioned intensity from SH sample, and lighter shading indicates RH samples. D . Boxplot of log 2 intensities for keratin 18 and keratin 8 protein levels in all three cell lines with significance as determined by Student’s t -test with multiple testing correction (* = 0.05, ** = 0.01). Each point indicates a value for either K1C18 (black) or K2C8 (grey) in each biological replicate. E . Immunofluorescence imaging with anti-keratin 18 (green) and anti-keratin 8 (red) antibodies in all cell lines indicating protein level differences consistent with proteome measurements.

    Techniques Used: Produced, Immunofluorescence, Imaging

    A, B . Boxplots of all ions quantified in MCF-7/HeLa reverse replicate (A) or HEK293/HeLa reverse replicate (B). C-F . Decomposition of observed isotopic envelops of released peptides with iqPIR algorithm into each channel (RH or SH) contribution. G . Reporter spectra with light reporter (808) in yellow and heavy reporter (812) in orange. Ratios of the reporter channels is closer to 1 than for peptides and their fragments because of ratio compression.
    Figure Legend Snippet: A, B . Boxplots of all ions quantified in MCF-7/HeLa reverse replicate (A) or HEK293/HeLa reverse replicate (B). C-F . Decomposition of observed isotopic envelops of released peptides with iqPIR algorithm into each channel (RH or SH) contribution. G . Reporter spectra with light reporter (808) in yellow and heavy reporter (812) in orange. Ratios of the reporter channels is closer to 1 than for peptides and their fragments because of ratio compression.

    Techniques Used:

    A . Cytoscape network for hnRNPs. Each node represents cross-linked residue with its number indicated inside the node. Edges represent detected and quantified cross- links. B . Boxplots comparing DIA based protein levels for hnRNPs in three cell lines. C . Heatmap showing log 2 fold change in HEK293 or MCF-7 compared to HeLa of protein levels based on DIA measurements for hnRNPs. D . Distributions of average log 2 fold changes for all cross-links (left) and proteins (right).
    Figure Legend Snippet: A . Cytoscape network for hnRNPs. Each node represents cross-linked residue with its number indicated inside the node. Edges represent detected and quantified cross- links. B . Boxplots comparing DIA based protein levels for hnRNPs in three cell lines. C . Heatmap showing log 2 fold change in HEK293 or MCF-7 compared to HeLa of protein levels based on DIA measurements for hnRNPs. D . Distributions of average log 2 fold changes for all cross-links (left) and proteins (right).

    Techniques Used: Residue

    A. Distributions of average log 2 ratios of RNA binding proteins on a cross-link level (left) and protein level (right). B. Boxplots of log 2 intensities of HNRPC and ROA1 protein levels in all three cell lines with significance, as determined by Student’s t -test with multiple testing correction (** = 0.01, ns = non-significant). C . Cross-links for HNRNPC, mapped to the SFPQ crystal structure (PDB 4WIJ). Cross-links from HEK293/HeLa and MCF-7/HeLa are indicated with thick transparent lines and thin solid lines, respectively. The log 2 ratio intensities are represented in green for decreasing ratios compared to control, in red for increasing ratios, or black for non- changing ratios. D. Cross-linked peptides for ROA1 mapped to a previously described modeled structure . Lysine K350, indicated in blue, is part of the nuclear targeting sequence M9.
    Figure Legend Snippet: A. Distributions of average log 2 ratios of RNA binding proteins on a cross-link level (left) and protein level (right). B. Boxplots of log 2 intensities of HNRPC and ROA1 protein levels in all three cell lines with significance, as determined by Student’s t -test with multiple testing correction (** = 0.01, ns = non-significant). C . Cross-links for HNRNPC, mapped to the SFPQ crystal structure (PDB 4WIJ). Cross-links from HEK293/HeLa and MCF-7/HeLa are indicated with thick transparent lines and thin solid lines, respectively. The log 2 ratio intensities are represented in green for decreasing ratios compared to control, in red for increasing ratios, or black for non- changing ratios. D. Cross-linked peptides for ROA1 mapped to a previously described modeled structure . Lysine K350, indicated in blue, is part of the nuclear targeting sequence M9.

    Techniques Used: RNA Binding Assay, Sequencing

    A. Heatmap of log 2 ratio of SMCA5 K847-K855 intra-link. B . Boxplot of SMCA5 log 2 intensities in HEK293, MCF-7 and HeLa with significance as determined by Student’s t -test with multiple testing correction (ns = non-significant). C . SMCA5 cross-link mapped to the structure of fly SANT domain (PDB:1OFC) (left) and cross-linked residues (magenta) mapped on the structure of yeast ISWE1a complexed with dinucleosomes (PDB:7X3T) (right).
    Figure Legend Snippet: A. Heatmap of log 2 ratio of SMCA5 K847-K855 intra-link. B . Boxplot of SMCA5 log 2 intensities in HEK293, MCF-7 and HeLa with significance as determined by Student’s t -test with multiple testing correction (ns = non-significant). C . SMCA5 cross-link mapped to the structure of fly SANT domain (PDB:1OFC) (left) and cross-linked residues (magenta) mapped on the structure of yeast ISWE1a complexed with dinucleosomes (PDB:7X3T) (right).

    Techniques Used:

    A. Heatmap of log 2 ratios of LGUL K148-K157 cross-links in HEK293/HeLa and MCF-7/HeLa. B. Boxplot of LGUL log 2 protein intensities in HEK293, MCF-7, and HeLa with significance as determined by Student’s t -test with multiple testing correction (* = 0.05, ns = non-significant). C. LGUL K148-K157 cross-link mapped to a human structure with an S-benzyl-glutathione inhibitor (located between cross-linked residues) (1FRO). Cysteine modified by inhibitory glutathionylation is indicated in yellow.
    Figure Legend Snippet: A. Heatmap of log 2 ratios of LGUL K148-K157 cross-links in HEK293/HeLa and MCF-7/HeLa. B. Boxplot of LGUL log 2 protein intensities in HEK293, MCF-7, and HeLa with significance as determined by Student’s t -test with multiple testing correction (* = 0.05, ns = non-significant). C. LGUL K148-K157 cross-link mapped to a human structure with an S-benzyl-glutathione inhibitor (located between cross-linked residues) (1FRO). Cysteine modified by inhibitory glutathionylation is indicated in yellow.

    Techniques Used: Modification

    A . Heatmap of log 2 ratios of ADT2 and ADT3 cross-links for MCF-7/HeLa (top) and HEK293/HeLa (bottom). B. Boxplot of ADT2 and ADT3 log 2 intensities in HEK293, MCF-7, and HeLa with significance as determined by Student’s t-test with multiple testing correction (* = 0.05, ns = non-significant). Right: View from the mitochondrial matrix side. C . K147-K272 and K147-K33 cross-links mapped on a c-state structure (PDB: 2C3E) and m-state structure (PDB: 6GCI). K33-K147 cross-link is mapped with Euclidean distance as SASD calculated values exceed 99 Å. D . Heatmap of log 2 ratios in c2c12 isolated mitochondria treated with m-state inhibitor (BKA) or c-state inhibitor (CATR).
    Figure Legend Snippet: A . Heatmap of log 2 ratios of ADT2 and ADT3 cross-links for MCF-7/HeLa (top) and HEK293/HeLa (bottom). B. Boxplot of ADT2 and ADT3 log 2 intensities in HEK293, MCF-7, and HeLa with significance as determined by Student’s t-test with multiple testing correction (* = 0.05, ns = non-significant). Right: View from the mitochondrial matrix side. C . K147-K272 and K147-K33 cross-links mapped on a c-state structure (PDB: 2C3E) and m-state structure (PDB: 6GCI). K33-K147 cross-link is mapped with Euclidean distance as SASD calculated values exceed 99 Å. D . Heatmap of log 2 ratios in c2c12 isolated mitochondria treated with m-state inhibitor (BKA) or c-state inhibitor (CATR).

    Techniques Used: Isolation

    hek293 cells  (ATCC)


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    ATCC hek293 cells
    MiSeq analysis was performed on <t>HEK293</t> cells treated with combinations of base editors (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) and guide RNAs (gRNAs) to determine the proportion of the sequence reads containing indels. Replicate samples for HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for gRNA. Each box shows the maximum, upper quarter, median, lower quarter, and minimum (from the top) based on three independent transfection experiments. Red circles represent individual data points. None was significant compared to corresponding EV-treated cells by Bonferroni-corrected Student’s t-test (eight tests for each base editor).
    Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington’s disease"

    Article Title: Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington’s disease

    Journal: eLife

    doi: 10.7554/eLife.89782

    MiSeq analysis was performed on HEK293 cells treated with combinations of base editors (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) and guide RNAs (gRNAs) to determine the proportion of the sequence reads containing indels. Replicate samples for HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for gRNA. Each box shows the maximum, upper quarter, median, lower quarter, and minimum (from the top) based on three independent transfection experiments. Red circles represent individual data points. None was significant compared to corresponding EV-treated cells by Bonferroni-corrected Student’s t-test (eight tests for each base editor).
    Figure Legend Snippet: MiSeq analysis was performed on HEK293 cells treated with combinations of base editors (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) and guide RNAs (gRNAs) to determine the proportion of the sequence reads containing indels. Replicate samples for HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for gRNA. Each box shows the maximum, upper quarter, median, lower quarter, and minimum (from the top) based on three independent transfection experiments. Red circles represent individual data points. None was significant compared to corresponding EV-treated cells by Bonferroni-corrected Student’s t-test (eight tests for each base editor).

    Techniques Used: Sequencing, Plasmid Preparation, Transfection

    Only CAG-to-CAA conversion showed significantly increased levels over the baseline sequencing errors. Thus, we calculated the percentage of CAA in the cells that were treated with a combination of cytosine base editors (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) and guide RNAs (gRNAs) (n=3). HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for gRNA. *, significant by Bonferroni-corrected p-value<0.05 (eight tests for each base editor).
    Figure Legend Snippet: Only CAG-to-CAA conversion showed significantly increased levels over the baseline sequencing errors. Thus, we calculated the percentage of CAA in the cells that were treated with a combination of cytosine base editors (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) and guide RNAs (gRNAs) (n=3). HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for gRNA. *, significant by Bonferroni-corrected p-value<0.05 (eight tests for each base editor).

    Techniques Used: Sequencing, Plasmid Preparation

    We calculated the mean percentage of sequence reads containing CAA at specific sites relative to all sequence reads (n=3). For example, 27.7% conversion at the 2nd CAG by BE4max-gRNA 1 (top left panel, red) means 27.7% of all sequence reads in HEK293 cells have CAA at the 2nd CAG. X-axis and Y-axis represent the position of the CAG and percent conversion. Each panel represents a tested gRNA. Plots were based on the mean of three independent transfection experiments in HEK293 cells after subtracting corresponding empty vector (EV)-treated cell data. Red, blue, purple, and cyan traces represent BE4max, BE4-NG, BE4-SpG, and evo-SpG, respectively.
    Figure Legend Snippet: We calculated the mean percentage of sequence reads containing CAA at specific sites relative to all sequence reads (n=3). For example, 27.7% conversion at the 2nd CAG by BE4max-gRNA 1 (top left panel, red) means 27.7% of all sequence reads in HEK293 cells have CAA at the 2nd CAG. X-axis and Y-axis represent the position of the CAG and percent conversion. Each panel represents a tested gRNA. Plots were based on the mean of three independent transfection experiments in HEK293 cells after subtracting corresponding empty vector (EV)-treated cell data. Red, blue, purple, and cyan traces represent BE4max, BE4-NG, BE4-SpG, and evo-SpG, respectively.

    Techniques Used: Sequencing, Transfection, Plasmid Preparation

    On the top, an example of duplicated interruption is displayed; filled red and orange in the example diagram represent original CAA interruption and CAA produced by BE strategies, respectively. The proportion of duplicated interruption in HEK293 cells treated with different BE strategies are displayed (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) (n=3 independent experiments). HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for guide RNA (gRNA). *, significant by Bonferroni-corrected p-value<0.05 (eight tests for each base editor).
    Figure Legend Snippet: On the top, an example of duplicated interruption is displayed; filled red and orange in the example diagram represent original CAA interruption and CAA produced by BE strategies, respectively. The proportion of duplicated interruption in HEK293 cells treated with different BE strategies are displayed (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) (n=3 independent experiments). HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for guide RNA (gRNA). *, significant by Bonferroni-corrected p-value<0.05 (eight tests for each base editor).

    Techniques Used: Produced, Plasmid Preparation

    On the top, an example of duplicated interruption with additional CAG-to-CAA conversion is displayed; filled red and orange in the example diagram represent original CAA interruption and new CAA produced by base editing (BE) strategies, respectively. The proportion of sequence reads containing duplicated interruption and CAG-to-CAA conversions at other sites in HEK293 cells treated with different BE strategies are displayed (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) (n=3 independent experiments). An example of such modification is displayed at the top. HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for guide RNA (gRNA). *, significant by Bonferroni-corrected p-value<0.05 (eight tests for each base editor).
    Figure Legend Snippet: On the top, an example of duplicated interruption with additional CAG-to-CAA conversion is displayed; filled red and orange in the example diagram represent original CAA interruption and new CAA produced by base editing (BE) strategies, respectively. The proportion of sequence reads containing duplicated interruption and CAG-to-CAA conversions at other sites in HEK293 cells treated with different BE strategies are displayed (A, BE4max; B, BE4-NG; C, BE4-SpG; and D, evo-SpG) (n=3 independent experiments). An example of such modification is displayed at the top. HEK293 cells without any treatment (i.e. Cell) were combined (n=8) and plotted for each base editor. EV represents HEK293 cells treated with a base editor and empty vector for guide RNA (gRNA). *, significant by Bonferroni-corrected p-value<0.05 (eight tests for each base editor).

    Techniques Used: Produced, Sequencing, Modification, Plasmid Preparation

    To determine whether different conversion efficiencies and patterns were due to transfection efficiencies, we treated HEK293 cells with guide RNA (gRNA) 2, and determined transfection efficiencies ( A ) and conversion efficiencies ( B ). We compared transfection efficiency (X-axis) with the conversion efficiencies at the 4th CAG (Y-axis in the panel B). Plots were based on the mean of three independent transfection experiments.
    Figure Legend Snippet: To determine whether different conversion efficiencies and patterns were due to transfection efficiencies, we treated HEK293 cells with guide RNA (gRNA) 2, and determined transfection efficiencies ( A ) and conversion efficiencies ( B ). We compared transfection efficiency (X-axis) with the conversion efficiencies at the 4th CAG (Y-axis in the panel B). Plots were based on the mean of three independent transfection experiments.

    Techniques Used: Transfection

    The sequence reads in our MiSeq data with at least one CAG-to-CAA conversion were analyzed to count the number of CAG-to-CAA conversions in a given sequence read. The proportion was calculated by subtracting corresponding empty vector (EV)-treated cell data (Y-axis), meaning the percentage of sequence reads containing a given number of multiple conversions relative to all sequence reads from the original 16 or 17 CAG alleles. Red, blue, purple, and cyan traces represent BE4max, BE4-NG, BE4-SpG, and evo-SpG, respectively. Each panel shows a tested guide RNA (gRNA); plots were based on the mean of three independent transfection experiments in HEK293 cells.
    Figure Legend Snippet: The sequence reads in our MiSeq data with at least one CAG-to-CAA conversion were analyzed to count the number of CAG-to-CAA conversions in a given sequence read. The proportion was calculated by subtracting corresponding empty vector (EV)-treated cell data (Y-axis), meaning the percentage of sequence reads containing a given number of multiple conversions relative to all sequence reads from the original 16 or 17 CAG alleles. Red, blue, purple, and cyan traces represent BE4max, BE4-NG, BE4-SpG, and evo-SpG, respectively. Each panel shows a tested guide RNA (gRNA); plots were based on the mean of three independent transfection experiments in HEK293 cells.

    Techniques Used: Sequencing, Plasmid Preparation, Transfection

    To determine the levels of CAG-to-CAA conversions in the CAG repeats of other polyglutamine disease genes (A, ATXN1 ; B, ATXN2 ; C, ATXN3 ; D, CACNA1A ; E, ATXN7 ; F, TBP ; G, ATN1 ; and H, AR ), we further analyzed representative HEK293 cells treated with base editing (BE) strategies (n=1). EV represents empty vector-treated cells. Red, blue, purple, and cyan traces represent BE4max, BE4-NG, BE4-SpG, and evo-SpG, respectively.
    Figure Legend Snippet: To determine the levels of CAG-to-CAA conversions in the CAG repeats of other polyglutamine disease genes (A, ATXN1 ; B, ATXN2 ; C, ATXN3 ; D, CACNA1A ; E, ATXN7 ; F, TBP ; G, ATN1 ; and H, AR ), we further analyzed representative HEK293 cells treated with base editing (BE) strategies (n=1). EV represents empty vector-treated cells. Red, blue, purple, and cyan traces represent BE4max, BE4-NG, BE4-SpG, and evo-SpG, respectively.

    Techniques Used: Plasmid Preparation

    The HEK293-51 CAG cell line was generated by knocking in a 51 CAG canonical repeat using CRISPR-Cas9. To validate the correct replacement of repeats, we analyzed DNA, RNA, and protein samples. ( A ) HTT CAG repeat region was amplified to detect the presence of the expanded CAG repeat in the DNA. ( B ) Similarly, RNA samples were amplified focusing on the CAG repeat region to determine whether HEK293-51 CAG cells express HTT mRNA harboring an expanded repeat. ( C ) Immunoblot analysis was performed to confirm the expression of mutant HTT protein. Note, the separation between mutant and normal HTT protein was marginal because the size difference between the two is relatively very small ( C ). HEK293 and HEK293-51 CAG represent original carrying 16/17 CAG canonical repeats and HEK293 cells carrying 17/51 CAG canonical repeats, respectively. Figure 5—figure supplement 1—source data 1. Unedited original images of . Figure 5—figure supplement 1—source data 2. Uncropped images with labels of .
    Figure Legend Snippet: The HEK293-51 CAG cell line was generated by knocking in a 51 CAG canonical repeat using CRISPR-Cas9. To validate the correct replacement of repeats, we analyzed DNA, RNA, and protein samples. ( A ) HTT CAG repeat region was amplified to detect the presence of the expanded CAG repeat in the DNA. ( B ) Similarly, RNA samples were amplified focusing on the CAG repeat region to determine whether HEK293-51 CAG cells express HTT mRNA harboring an expanded repeat. ( C ) Immunoblot analysis was performed to confirm the expression of mutant HTT protein. Note, the separation between mutant and normal HTT protein was marginal because the size difference between the two is relatively very small ( C ). HEK293 and HEK293-51 CAG represent original carrying 16/17 CAG canonical repeats and HEK293 cells carrying 17/51 CAG canonical repeats, respectively. Figure 5—figure supplement 1—source data 1. Unedited original images of . Figure 5—figure supplement 1—source data 2. Uncropped images with labels of .

    Techniques Used: Generated, CRISPR, Amplification, Western Blot, Expressing, Mutagenesis

    ( A ) To overcome the limitations of patient-derived induced pluripotent stem cell (iPSC) and differentiated neurons, we developed HEK293 carrying an adult-onset CAG repeat by replacing one of the normal repeats with 51 canonical CAG (namely HEK293-51 CAG). Red and green bars represent respectively mutant and normal HTT in HEK293-51 CAG cells. ( B and C ) The HEK293-51 CAG cells were treated with BE4max-gRNA 1 and analyzed to determine the levels of in-frame insertion ( B ) and in-frame deletion ( C ) at the time of treatment (n=4). ( D ) The HEK293-51 CAG cells were treated with the gRNA 1 and analyzed by MiSeq to determine the levels of allele specificity. Conversion efficiency on the Y-axis indicates the percentage of sequence reads containing the CAG-to-CAA conversion at the target site (n=3). * represents uncorrected p-value<0.05 by Student’s t-test. ( E ) Original HEK293 cells and HEK293-51 CAG cells were treated with empty vector (EV), or candidate BE strategies (BE4max-gRNA 1 and BE4max-gRNA 2) and subjected to immunoblot analysis; representative blot is shown in panel E (n=3). ( F ) Four independent experiments were performed, and we performed one-sample t-test to determine whether BE-treated cells show different total HTT protein levels compared to EV-treated cells (n=4). Nothing was significant by p-value<0.05. Figure 5—source data 1. Unedited original images of the western blot analysis in . Figure 5—source data 2. Uncropped images with labels of the western blot analysis in .
    Figure Legend Snippet: ( A ) To overcome the limitations of patient-derived induced pluripotent stem cell (iPSC) and differentiated neurons, we developed HEK293 carrying an adult-onset CAG repeat by replacing one of the normal repeats with 51 canonical CAG (namely HEK293-51 CAG). Red and green bars represent respectively mutant and normal HTT in HEK293-51 CAG cells. ( B and C ) The HEK293-51 CAG cells were treated with BE4max-gRNA 1 and analyzed to determine the levels of in-frame insertion ( B ) and in-frame deletion ( C ) at the time of treatment (n=4). ( D ) The HEK293-51 CAG cells were treated with the gRNA 1 and analyzed by MiSeq to determine the levels of allele specificity. Conversion efficiency on the Y-axis indicates the percentage of sequence reads containing the CAG-to-CAA conversion at the target site (n=3). * represents uncorrected p-value<0.05 by Student’s t-test. ( E ) Original HEK293 cells and HEK293-51 CAG cells were treated with empty vector (EV), or candidate BE strategies (BE4max-gRNA 1 and BE4max-gRNA 2) and subjected to immunoblot analysis; representative blot is shown in panel E (n=3). ( F ) Four independent experiments were performed, and we performed one-sample t-test to determine whether BE-treated cells show different total HTT protein levels compared to EV-treated cells (n=4). Nothing was significant by p-value<0.05. Figure 5—source data 1. Unedited original images of the western blot analysis in . Figure 5—source data 2. Uncropped images with labels of the western blot analysis in .

    Techniques Used: Derivative Assay, Mutagenesis, Sequencing, Plasmid Preparation, Western Blot

    ( A ) HEK293 cells were treated with empty vector (EV) or candidate BE strategies such as BE4max-gRNA 1 (gRNA 1) and BE4max-gRNA 2 (gRNA 2) for RNAseq analysis. MiSeq analysis was also performed to judge the levels of CAG-to-CAA conversion. ****, p-value<0.0001 by Student’s t-test (n=4). ( B ) Confirming the lack of significantly altered genes in BE4max-gRNA 1 or BE4max-gRNA 2, we compared all BE-treated samples (n=8) with all EV-treated samples (n=4) to increase the power in the RNAseq differential gene expression analysis. Each circle in the volcano plot represents a gene analyzed in the RNAseq; HTT is indicated by a filled red circle. A red horizontal line represents false discovery rate of 0.05, showing that none was significantly altered by candidate BE strategies. ( C ) We also compared two groups of randomly assigned samples (six samples vs. six samples) to understand the shape of the volcano plot when there were no significant genes.
    Figure Legend Snippet: ( A ) HEK293 cells were treated with empty vector (EV) or candidate BE strategies such as BE4max-gRNA 1 (gRNA 1) and BE4max-gRNA 2 (gRNA 2) for RNAseq analysis. MiSeq analysis was also performed to judge the levels of CAG-to-CAA conversion. ****, p-value<0.0001 by Student’s t-test (n=4). ( B ) Confirming the lack of significantly altered genes in BE4max-gRNA 1 or BE4max-gRNA 2, we compared all BE-treated samples (n=8) with all EV-treated samples (n=4) to increase the power in the RNAseq differential gene expression analysis. Each circle in the volcano plot represents a gene analyzed in the RNAseq; HTT is indicated by a filled red circle. A red horizontal line represents false discovery rate of 0.05, showing that none was significantly altered by candidate BE strategies. ( C ) We also compared two groups of randomly assigned samples (six samples vs. six samples) to understand the shape of the volcano plot when there were no significant genes.

    Techniques Used: Plasmid Preparation, Expressing

    HEK293 cells were treated with empty vector (EV), or candidate base editing (BE) strategies such as BE4max-gRNA 1 ( A ) and BE4max-gRNA 2 ( B ). Subsequently, DNA samples and RNA samples were collected for MiSeq analysis and RNAseq analysis to evaluate the levels of on-target conversion and changes in transcriptome (n=4), respectively. The most significant gene in cells treated with BE4max-gRNA 2 (panel B) was HSD3B1 , which was not significant by false discovery rate of 0.05 (red lines).
    Figure Legend Snippet: HEK293 cells were treated with empty vector (EV), or candidate base editing (BE) strategies such as BE4max-gRNA 1 ( A ) and BE4max-gRNA 2 ( B ). Subsequently, DNA samples and RNA samples were collected for MiSeq analysis and RNAseq analysis to evaluate the levels of on-target conversion and changes in transcriptome (n=4), respectively. The most significant gene in cells treated with BE4max-gRNA 2 (panel B) was HSD3B1 , which was not significant by false discovery rate of 0.05 (red lines).

    Techniques Used: Plasmid Preparation

    hek293 cells  (ATCC)


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    ATCC hek293 cells
    Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hek293 cells
    (A) Cytotoxicity (measured by LDH release) of combinations of three constructs transfected in <t>HEK293T</t> cells. Each data point is a biological replicate, normalized to LDH release from transfection with peel-1 ::eGFP in the same experiment. All plots show means with SD. Transfections combine constructs encoding for mCherry or eGFP (-) or a fluorescent-tagged protein (+): PEEL-1::eGFP (top), PMPL-1::mCherry (middle), or mCherry::ZEEL-1 (bottom). (B) Cytotoxicity of PMP3-like proteins alone or with PEEL-1::eGFP. The PMPL-1 yak52 (A47T) mutant protein, C. elegans PMPL-2, and the yeast homolog yPMP3 are shown. (C) Live-cell imaging of a single cell transfected with an ER-marker (mCherry::KDEL) and peel-1 ::eGFP or (D) pmpl-1 ::eGFP. The cell nucleus is indicated (N). PMPL-1 is also seen on the plasma membrane (PM) and on lipid droplets (inset). Scale bar = 10μm. (E) Cytotoxicity is suppressed by addition of the GBR1, ER-retention tag on the C-terminus of PEEL-1::eGFP or PMPL-1::mCherry. P-values in (A) and (E) calculated using one-way ANOVA with Dunnett’s multiple comparisons test, comparing all samples to PEEL-1 alone in (A) and all samples to PEEL-1 with PMPL-1 in (E). In (B), multiple unpaired t-tests were used with Holm-Šídák test, comparing each PMP3-like protein alone to PMP3-like with PEEL-1 (***, p < 0.001; ****, p < 0.0001).
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    1) Product Images from "Mechanism of an animal toxin-antidote system"

    Article Title: Mechanism of an animal toxin-antidote system

    Journal: bioRxiv

    doi: 10.1101/2024.06.11.598564

    (A) Cytotoxicity (measured by LDH release) of combinations of three constructs transfected in HEK293T cells. Each data point is a biological replicate, normalized to LDH release from transfection with peel-1 ::eGFP in the same experiment. All plots show means with SD. Transfections combine constructs encoding for mCherry or eGFP (-) or a fluorescent-tagged protein (+): PEEL-1::eGFP (top), PMPL-1::mCherry (middle), or mCherry::ZEEL-1 (bottom). (B) Cytotoxicity of PMP3-like proteins alone or with PEEL-1::eGFP. The PMPL-1 yak52 (A47T) mutant protein, C. elegans PMPL-2, and the yeast homolog yPMP3 are shown. (C) Live-cell imaging of a single cell transfected with an ER-marker (mCherry::KDEL) and peel-1 ::eGFP or (D) pmpl-1 ::eGFP. The cell nucleus is indicated (N). PMPL-1 is also seen on the plasma membrane (PM) and on lipid droplets (inset). Scale bar = 10μm. (E) Cytotoxicity is suppressed by addition of the GBR1, ER-retention tag on the C-terminus of PEEL-1::eGFP or PMPL-1::mCherry. P-values in (A) and (E) calculated using one-way ANOVA with Dunnett’s multiple comparisons test, comparing all samples to PEEL-1 alone in (A) and all samples to PEEL-1 with PMPL-1 in (E). In (B), multiple unpaired t-tests were used with Holm-Šídák test, comparing each PMP3-like protein alone to PMP3-like with PEEL-1 (***, p < 0.001; ****, p < 0.0001).
    Figure Legend Snippet: (A) Cytotoxicity (measured by LDH release) of combinations of three constructs transfected in HEK293T cells. Each data point is a biological replicate, normalized to LDH release from transfection with peel-1 ::eGFP in the same experiment. All plots show means with SD. Transfections combine constructs encoding for mCherry or eGFP (-) or a fluorescent-tagged protein (+): PEEL-1::eGFP (top), PMPL-1::mCherry (middle), or mCherry::ZEEL-1 (bottom). (B) Cytotoxicity of PMP3-like proteins alone or with PEEL-1::eGFP. The PMPL-1 yak52 (A47T) mutant protein, C. elegans PMPL-2, and the yeast homolog yPMP3 are shown. (C) Live-cell imaging of a single cell transfected with an ER-marker (mCherry::KDEL) and peel-1 ::eGFP or (D) pmpl-1 ::eGFP. The cell nucleus is indicated (N). PMPL-1 is also seen on the plasma membrane (PM) and on lipid droplets (inset). Scale bar = 10μm. (E) Cytotoxicity is suppressed by addition of the GBR1, ER-retention tag on the C-terminus of PEEL-1::eGFP or PMPL-1::mCherry. P-values in (A) and (E) calculated using one-way ANOVA with Dunnett’s multiple comparisons test, comparing all samples to PEEL-1 alone in (A) and all samples to PEEL-1 with PMPL-1 in (E). In (B), multiple unpaired t-tests were used with Holm-Šídák test, comparing each PMP3-like protein alone to PMP3-like with PEEL-1 (***, p < 0.001; ****, p < 0.0001).

    Techniques Used: Construct, Transfection, Mutagenesis, Live Cell Imaging, Marker, Membrane

    (A) The AlphaFold2 predicted structure of PEEL-1 and (B) a helical wheel representation of the putative PEEL-1 amphipathic helix. Amphipathic helix residues are colored (pink = hydrophobic, blue = hydrophilic). (C) Cytotoxicity of a series of PEEL-1 C-terminal truncations expressed in HEK293T cells. The number of amino acids removed are indicated (ex. “-28” means the last 28 residues were removed). Each truncation removes an additional alpha helix. The “-65” truncation removes the amphipathic helix. (D) Percent dead worms after heat-shock PEEL-1 expression of the indicated truncation mutant. 50 worms were assayed for each data point, and two independent transgenic lines were tested for each construct. (E) Cytotoxicity of PEEL-1 amphipathic helix missense mutants in HEK293T cells. Mutants are ordered by descending hydrophobic moment (μH). Six single mutants (left of dotted line) and three double mutants are shown (right of dotted line). All bar graphs show mean with SD. Statistics were performed using multiple unpaired t-tests with Holm-Šídák test, comparing each PEEL-1 alone to PEEL-1 and PMPL-1 (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001). (F) The average toxicity of missense mutants from (E) plotted against their hydrophobic moment.
    Figure Legend Snippet: (A) The AlphaFold2 predicted structure of PEEL-1 and (B) a helical wheel representation of the putative PEEL-1 amphipathic helix. Amphipathic helix residues are colored (pink = hydrophobic, blue = hydrophilic). (C) Cytotoxicity of a series of PEEL-1 C-terminal truncations expressed in HEK293T cells. The number of amino acids removed are indicated (ex. “-28” means the last 28 residues were removed). Each truncation removes an additional alpha helix. The “-65” truncation removes the amphipathic helix. (D) Percent dead worms after heat-shock PEEL-1 expression of the indicated truncation mutant. 50 worms were assayed for each data point, and two independent transgenic lines were tested for each construct. (E) Cytotoxicity of PEEL-1 amphipathic helix missense mutants in HEK293T cells. Mutants are ordered by descending hydrophobic moment (μH). Six single mutants (left of dotted line) and three double mutants are shown (right of dotted line). All bar graphs show mean with SD. Statistics were performed using multiple unpaired t-tests with Holm-Šídák test, comparing each PEEL-1 alone to PEEL-1 and PMPL-1 (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001). (F) The average toxicity of missense mutants from (E) plotted against their hydrophobic moment.

    Techniques Used: Expressing, Mutagenesis, Transgenic Assay, Construct

    Current-voltage plots of whole-cell patch-clamp electrophysiology on (A) transfected cells, (B) Control cell line, and (C) Experimental cell line. High intracellular potassium (140 mM K + / 8.6 mM Na + ) and high extracellular sodium (145 mM Na + / 4 mM K + ) solutions are used. Currents elicited by a family of 0.5 second voltage steps from a -30 mV holding potential, from -100 mV to 60 mV, in 10 mV increments. Currents normalized to cell capacitance (pF). Negative and positive currents indicate cation flow into or out of the cell, respectively. (A) Plots from HEK293 cells acutely transfected with peel-1 ::eGFP or pmpl-1 ::mCherry. (B) Plots from Control cell line expressing constitutive eGFP and tetracycline-inducible pmpl-1 ::mCherry. (C) Plots from Experimental cell line expressing constitutive peel-1 ::eGFP and tetracycline-inducible pmpl-1 ::mCherry. Control and Experimental cell lines are shown without tetracycline or with tetracycline at the indicated time after addition of tetracycline. All plots shown as mean with SEM. (D) Western blot for PMPL-1::mCherry in Control and Experimental cell lines without tetracycline (-tet, left) and 18 hours after addition of tetracycline (+tet, right). Leaky expression of PMPL-1 is seen in both cell lines in the absence of tetracycline. Less background PMPL-1 expression is seen in Experimental cells than in Control cells, likely because of selection against higher background PMPL-1 expression when in combination with PEEL-1 but not eGFP. GAPDH loading control shown. (E) Permeability of indicated ions was assayed in Experimental cells without tetracycline. Test ionic solutions substituted previous bath solution (145 mM Na + / 4 mM K + ) with 140 mM pure cations (external) or 20 mM anions (internal), except Ca 2+ (20 mM external, 1 mM internal Ca 2+ with 2.5 mM EGTA). All plots show mean with SEM. Statistical tests compare all results to NMDG (treated as control) in one-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05; ***, p < 0.001). (F) Schematic of planar lipid bilayer experiment. Liposomes containing purified PEEL-1 or PMPL-1 are added to the cis well to deliver proteins to the lipid bilayer. (G) Conductance traces of one experiment (left) and a histogram of the trace (right, 2 pS bin width, normalized based on probability density) at an applied voltage of +180 mV. SDS-PAGE gels of purified proteins are shown in fig. S15A and more example conductance traces and controls are shown in fig. S16.
    Figure Legend Snippet: Current-voltage plots of whole-cell patch-clamp electrophysiology on (A) transfected cells, (B) Control cell line, and (C) Experimental cell line. High intracellular potassium (140 mM K + / 8.6 mM Na + ) and high extracellular sodium (145 mM Na + / 4 mM K + ) solutions are used. Currents elicited by a family of 0.5 second voltage steps from a -30 mV holding potential, from -100 mV to 60 mV, in 10 mV increments. Currents normalized to cell capacitance (pF). Negative and positive currents indicate cation flow into or out of the cell, respectively. (A) Plots from HEK293 cells acutely transfected with peel-1 ::eGFP or pmpl-1 ::mCherry. (B) Plots from Control cell line expressing constitutive eGFP and tetracycline-inducible pmpl-1 ::mCherry. (C) Plots from Experimental cell line expressing constitutive peel-1 ::eGFP and tetracycline-inducible pmpl-1 ::mCherry. Control and Experimental cell lines are shown without tetracycline or with tetracycline at the indicated time after addition of tetracycline. All plots shown as mean with SEM. (D) Western blot for PMPL-1::mCherry in Control and Experimental cell lines without tetracycline (-tet, left) and 18 hours after addition of tetracycline (+tet, right). Leaky expression of PMPL-1 is seen in both cell lines in the absence of tetracycline. Less background PMPL-1 expression is seen in Experimental cells than in Control cells, likely because of selection against higher background PMPL-1 expression when in combination with PEEL-1 but not eGFP. GAPDH loading control shown. (E) Permeability of indicated ions was assayed in Experimental cells without tetracycline. Test ionic solutions substituted previous bath solution (145 mM Na + / 4 mM K + ) with 140 mM pure cations (external) or 20 mM anions (internal), except Ca 2+ (20 mM external, 1 mM internal Ca 2+ with 2.5 mM EGTA). All plots show mean with SEM. Statistical tests compare all results to NMDG (treated as control) in one-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05; ***, p < 0.001). (F) Schematic of planar lipid bilayer experiment. Liposomes containing purified PEEL-1 or PMPL-1 are added to the cis well to deliver proteins to the lipid bilayer. (G) Conductance traces of one experiment (left) and a histogram of the trace (right, 2 pS bin width, normalized based on probability density) at an applied voltage of +180 mV. SDS-PAGE gels of purified proteins are shown in fig. S15A and more example conductance traces and controls are shown in fig. S16.

    Techniques Used: Patch Clamp, Transfection, Expressing, Western Blot, Selection, Permeability, Liposomes, Purification, SDS Page

    hek293 cells  (ATCC)


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    ATCC hek293 cells
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    human embryonic kidney hek293 cells  (ATCC)


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    ATCC human embryonic kidney hek293 cells
    NaLTSA assay for SYK(WT)-NL with MRL-SYKi. ( A ) Melting curves with the addition of MRL-SYKi in dose–response format in <t>HEK293</t> cells. Data reported are an average from biological triplicates each with two technical replicates. The data was fitted with a Boltzmann Sigmoid equation and error bars indicate the standard deviation ( B ) Visualization of the shift from vehicle with 30 μM of MRL-SYKi from ( A ). (C ) Melting temperatures (T m ) at each concentration of MRL-SYKi and the corresponding change in melting temperature (ΔT m ). Data are reported as an average of the T m ± SEM.
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    1) Product Images from "Development of SYK NanoBRET Cellular Target Engagement Assays for Gain–of–Function Variants"

    Article Title: Development of SYK NanoBRET Cellular Target Engagement Assays for Gain–of–Function Variants

    Journal: bioRxiv

    doi: 10.1101/2024.06.12.598544

    NaLTSA assay for SYK(WT)-NL with MRL-SYKi. ( A ) Melting curves with the addition of MRL-SYKi in dose–response format in HEK293 cells. Data reported are an average from biological triplicates each with two technical replicates. The data was fitted with a Boltzmann Sigmoid equation and error bars indicate the standard deviation ( B ) Visualization of the shift from vehicle with 30 μM of MRL-SYKi from ( A ). (C ) Melting temperatures (T m ) at each concentration of MRL-SYKi and the corresponding change in melting temperature (ΔT m ). Data are reported as an average of the T m ± SEM.
    Figure Legend Snippet: NaLTSA assay for SYK(WT)-NL with MRL-SYKi. ( A ) Melting curves with the addition of MRL-SYKi in dose–response format in HEK293 cells. Data reported are an average from biological triplicates each with two technical replicates. The data was fitted with a Boltzmann Sigmoid equation and error bars indicate the standard deviation ( B ) Visualization of the shift from vehicle with 30 μM of MRL-SYKi from ( A ). (C ) Melting temperatures (T m ) at each concentration of MRL-SYKi and the corresponding change in melting temperature (ΔT m ). Data are reported as an average of the T m ± SEM.

    Techniques Used: Standard Deviation, Concentration Assay

    SYK inhibitors bind potently to SYK GoF variants, reducing SYK phosphorylation and SYK catalytic activity. ( A ) IC 50 data for SYK inhibitors versus SYK GoF variants in HEK293 cells with NanoBRET tracer 8 . Data are reported as an average of triplicate IC 50 data ± SEM. Data reported are from a single experiment and plotted using a log(inhibitor) vs. response (three-parameter) fit. ( B ) Quantitative analysis of BRET using Cheng-Prusoff relationship for SYK variants in HEK293 cells with NanoBRET tracer 8 . The apparent K D value for MRL-SYKi was determined from the y-intercept by linear regression. Data reported are from a single experiment and plotted in GraphPad Prism with a simple linear regression. ( C ) The kinase activity of SYK(S550F) and SYK(P342T) is inhibited with MRL-SYKi in the PhosphoSens Assay. ( D ) MRL-SYKi decreases pSYK(Y525/526) levels in a SW480 cell line stably expressing SYK(S550Y) after treatment at 100 nM for 20 hrs.
    Figure Legend Snippet: SYK inhibitors bind potently to SYK GoF variants, reducing SYK phosphorylation and SYK catalytic activity. ( A ) IC 50 data for SYK inhibitors versus SYK GoF variants in HEK293 cells with NanoBRET tracer 8 . Data are reported as an average of triplicate IC 50 data ± SEM. Data reported are from a single experiment and plotted using a log(inhibitor) vs. response (three-parameter) fit. ( B ) Quantitative analysis of BRET using Cheng-Prusoff relationship for SYK variants in HEK293 cells with NanoBRET tracer 8 . The apparent K D value for MRL-SYKi was determined from the y-intercept by linear regression. Data reported are from a single experiment and plotted in GraphPad Prism with a simple linear regression. ( C ) The kinase activity of SYK(S550F) and SYK(P342T) is inhibited with MRL-SYKi in the PhosphoSens Assay. ( D ) MRL-SYKi decreases pSYK(Y525/526) levels in a SW480 cell line stably expressing SYK(S550Y) after treatment at 100 nM for 20 hrs.

    Techniques Used: Activity Assay, Stable Transfection, Expressing

    hek 293 cells  (ATCC)


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    ATCC hek 293 cells
    a , Arrangement of the lux genes in the expression cassette. b , In the bioluminescence reaction, light is generated through the concomitant oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain aliphatic aldehyde (RCHO), catalyzed by the luciferase (LuxAB). The two products are recycled by the fatty acid reductase complex (LuxCDE) and a flavin reductase (Frp). c , Comparison of luminescence emission from <t>HEK-293</t> and HeLa cells transiently transfected with the expression cassette in a (1 plasmid) or a mixture of plasmids containing luxA, luxB, luxC, luxD, luxE and frp separately (6 plasmids). Plots show average values and standard deviations from 5 independent measurements.
    Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Autonomous bioluminescence emission from transgenic mice"

    Article Title: Autonomous bioluminescence emission from transgenic mice

    Journal: bioRxiv

    doi: 10.1101/2024.06.13.598801

    a , Arrangement of the lux genes in the expression cassette. b , In the bioluminescence reaction, light is generated through the concomitant oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain aliphatic aldehyde (RCHO), catalyzed by the luciferase (LuxAB). The two products are recycled by the fatty acid reductase complex (LuxCDE) and a flavin reductase (Frp). c , Comparison of luminescence emission from HEK-293 and HeLa cells transiently transfected with the expression cassette in a (1 plasmid) or a mixture of plasmids containing luxA, luxB, luxC, luxD, luxE and frp separately (6 plasmids). Plots show average values and standard deviations from 5 independent measurements.
    Figure Legend Snippet: a , Arrangement of the lux genes in the expression cassette. b , In the bioluminescence reaction, light is generated through the concomitant oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain aliphatic aldehyde (RCHO), catalyzed by the luciferase (LuxAB). The two products are recycled by the fatty acid reductase complex (LuxCDE) and a flavin reductase (Frp). c , Comparison of luminescence emission from HEK-293 and HeLa cells transiently transfected with the expression cassette in a (1 plasmid) or a mixture of plasmids containing luxA, luxB, luxC, luxD, luxE and frp separately (6 plasmids). Plots show average values and standard deviations from 5 independent measurements.

    Techniques Used: Expressing, Generated, Luciferase, Comparison, Transfection, Plasmid Preparation

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    ATCC human cell line hek293
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    ATCC hek293 cell lines
    A representative IV curves induced by the −100 to +100 mV voltage ramp recorded in <t>HEK</t> cells transfected with the vEF1ap-5’UTR-TRPV6wt_CMVp-GFP vector. Curves show whole-cell TRPV6 currents in either base HBSS/basal medium (black), DVF medium alone (red), or DVF medium containing either 12 µg/ml of mAb82 anti-TRPV6 antibody (pink) or 24 µg/ml (yellow). B representative trace of the whole-cell currents during the application of the DVF solution as well as different doses of mAb82 antibody, as indicated by arrows. C bar plots summarizing average whole-cell currents under conditions indicated above for mAb82, ( n = 15 for 1.2 µg/ml; n = 20 for 2.4 µg/ml; n = 12 for 6 µg/ml; n = 11 for 12 µg/ml; and n = 11 for 24 µg/ml). D SOCE in the LNCaP cells pretreated 5 min with either mouse monoclonal mAbAU1 as a control antibody or a mouse anti-human TRPV6 mAb82 antibody, both at 2.4 µg/ml. E corresponding quantitative representation of the ER content (calculated as a maximum amplitude), n = 3, * p < 0.05; the SOCE affected by antibody-induced treatments shown in ( D ); n = 3, *** p < 0.001; and the slope of SOCE calculated as a ratio delta/sec for each condition; n = 3, * p < 0.05.
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    ATCC hek293 cells
    Enzyme-mediated C-terminal cleavage exposes the CAD. (A) A schematic representation of the full length Panx1 harboring a TEV protease cleavage site instead of the caspase recognition site (Panx1-T). (B) and (C) Exemplar whole cell currents (B) and peak current density at +110 mV (C) of Panx1-T co-expressed in <t>HEK293</t> cells with TEV protease. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=4. Asterisks indicate significance of p<0.05 determined by student T-test. (D) Inside-out patch clamp recordings with or without recombinant TEV protease (0.1mg/mL). Voltage ramps from 130 mV to +80 mV over 0.5 s were applied every 6 s. Representative peak currents at +80 mV are plotted for Panx1-T (blue) and Panx1-T L367A/L370A (gray). CBX (100 μM) were applied at time points indicated by orange bars. (E) TEV specific currents defined as the peak current differences between the indicated time points a and b . N=4. Asterisk indicates a p<0.05 obtained from an unpaired student T-test.
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    ATCC human embryonic kidney hek293 cells
    NaLTSA assay for SYK(WT)-NL with MRL-SYKi. ( A ) Melting curves with the addition of MRL-SYKi in dose–response format in <t>HEK293</t> cells. Data reported are an average from biological triplicates each with two technical replicates. The data was fitted with a Boltzmann Sigmoid equation and error bars indicate the standard deviation ( B ) Visualization of the shift from vehicle with 30 μM of MRL-SYKi from ( A ). (C ) Melting temperatures (T m ) at each concentration of MRL-SYKi and the corresponding change in melting temperature (ΔT m ). Data are reported as an average of the T m ± SEM.
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    ATCC hek 293 cells
    a , Arrangement of the lux genes in the expression cassette. b , In the bioluminescence reaction, light is generated through the concomitant oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain aliphatic aldehyde (RCHO), catalyzed by the luciferase (LuxAB). The two products are recycled by the fatty acid reductase complex (LuxCDE) and a flavin reductase (Frp). c , Comparison of luminescence emission from <t>HEK-293</t> and HeLa cells transiently transfected with the expression cassette in a (1 plasmid) or a mixture of plasmids containing luxA, luxB, luxC, luxD, luxE and frp separately (6 plasmids). Plots show average values and standard deviations from 5 independent measurements.
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    A representative IV curves induced by the −100 to +100 mV voltage ramp recorded in HEK cells transfected with the vEF1ap-5’UTR-TRPV6wt_CMVp-GFP vector. Curves show whole-cell TRPV6 currents in either base HBSS/basal medium (black), DVF medium alone (red), or DVF medium containing either 12 µg/ml of mAb82 anti-TRPV6 antibody (pink) or 24 µg/ml (yellow). B representative trace of the whole-cell currents during the application of the DVF solution as well as different doses of mAb82 antibody, as indicated by arrows. C bar plots summarizing average whole-cell currents under conditions indicated above for mAb82, ( n = 15 for 1.2 µg/ml; n = 20 for 2.4 µg/ml; n = 12 for 6 µg/ml; n = 11 for 12 µg/ml; and n = 11 for 24 µg/ml). D SOCE in the LNCaP cells pretreated 5 min with either mouse monoclonal mAbAU1 as a control antibody or a mouse anti-human TRPV6 mAb82 antibody, both at 2.4 µg/ml. E corresponding quantitative representation of the ER content (calculated as a maximum amplitude), n = 3, * p < 0.05; the SOCE affected by antibody-induced treatments shown in ( D ); n = 3, *** p < 0.001; and the slope of SOCE calculated as a ratio delta/sec for each condition; n = 3, * p < 0.05.

    Journal: Cell Death & Disease

    Article Title: Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression

    doi: 10.1038/s41419-024-06809-0

    Figure Lengend Snippet: A representative IV curves induced by the −100 to +100 mV voltage ramp recorded in HEK cells transfected with the vEF1ap-5’UTR-TRPV6wt_CMVp-GFP vector. Curves show whole-cell TRPV6 currents in either base HBSS/basal medium (black), DVF medium alone (red), or DVF medium containing either 12 µg/ml of mAb82 anti-TRPV6 antibody (pink) or 24 µg/ml (yellow). B representative trace of the whole-cell currents during the application of the DVF solution as well as different doses of mAb82 antibody, as indicated by arrows. C bar plots summarizing average whole-cell currents under conditions indicated above for mAb82, ( n = 15 for 1.2 µg/ml; n = 20 for 2.4 µg/ml; n = 12 for 6 µg/ml; n = 11 for 12 µg/ml; and n = 11 for 24 µg/ml). D SOCE in the LNCaP cells pretreated 5 min with either mouse monoclonal mAbAU1 as a control antibody or a mouse anti-human TRPV6 mAb82 antibody, both at 2.4 µg/ml. E corresponding quantitative representation of the ER content (calculated as a maximum amplitude), n = 3, * p < 0.05; the SOCE affected by antibody-induced treatments shown in ( D ); n = 3, *** p < 0.001; and the slope of SOCE calculated as a ratio delta/sec for each condition; n = 3, * p < 0.05.

    Article Snippet: Human PC3M (metastatic cell line issued from PC3 cells grafted in vivo), LNCaP, and HEK293 cell lines were from American Type Culture Collection (ATCC) and were cultured in RPMI (LNCaP and PC3M), and DMEM (HEK293) media (Gibco-BRL, CergyPontoise, France) supplemented with 10% fetal calf serum and containing kanamycin (100 µg/mL) and L-glutamine (2 mM) where necessary.

    Techniques: Transfection, Plasmid Preparation

    Enzyme-mediated C-terminal cleavage exposes the CAD. (A) A schematic representation of the full length Panx1 harboring a TEV protease cleavage site instead of the caspase recognition site (Panx1-T). (B) and (C) Exemplar whole cell currents (B) and peak current density at +110 mV (C) of Panx1-T co-expressed in HEK293 cells with TEV protease. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=4. Asterisks indicate significance of p<0.05 determined by student T-test. (D) Inside-out patch clamp recordings with or without recombinant TEV protease (0.1mg/mL). Voltage ramps from 130 mV to +80 mV over 0.5 s were applied every 6 s. Representative peak currents at +80 mV are plotted for Panx1-T (blue) and Panx1-T L367A/L370A (gray). CBX (100 μM) were applied at time points indicated by orange bars. (E) TEV specific currents defined as the peak current differences between the indicated time points a and b . N=4. Asterisk indicates a p<0.05 obtained from an unpaired student T-test.

    Journal: bioRxiv

    Article Title: The C-terminal activating domain promotes Panx1 channel opening

    doi: 10.1101/2024.06.13.598903

    Figure Lengend Snippet: Enzyme-mediated C-terminal cleavage exposes the CAD. (A) A schematic representation of the full length Panx1 harboring a TEV protease cleavage site instead of the caspase recognition site (Panx1-T). (B) and (C) Exemplar whole cell currents (B) and peak current density at +110 mV (C) of Panx1-T co-expressed in HEK293 cells with TEV protease. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=4. Asterisks indicate significance of p<0.05 determined by student T-test. (D) Inside-out patch clamp recordings with or without recombinant TEV protease (0.1mg/mL). Voltage ramps from 130 mV to +80 mV over 0.5 s were applied every 6 s. Representative peak currents at +80 mV are plotted for Panx1-T (blue) and Panx1-T L367A/L370A (gray). CBX (100 μM) were applied at time points indicated by orange bars. (E) TEV specific currents defined as the peak current differences between the indicated time points a and b . N=4. Asterisk indicates a p<0.05 obtained from an unpaired student T-test.

    Article Snippet: HEK293 cells (ATCC: CRL-1573) were cultured in DMEM supplemented with 10% FBS at 37°C with 8% CO2 in a humidified incubator.

    Techniques: Patch Clamp, Recombinant

    CAD can facilitate Panx1 activation from non-native positions. Schematic representations ((A), (C), (F), and (H)), representative whole cell recordings ((B), (D), (G), (I)), and peak current density at +110 mV ((E) and (J)) are shown for each tested construct. HEK293 cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=5-24 and error bars indicate SEM. Asterisks indicate significance of p<0.05 determined by one-way ANOVA followed by Dunnett’s test comparing the Panx1Δ356 (E) or Panx1 wild type (J) to each construct.

    Journal: bioRxiv

    Article Title: The C-terminal activating domain promotes Panx1 channel opening

    doi: 10.1101/2024.06.13.598903

    Figure Lengend Snippet: CAD can facilitate Panx1 activation from non-native positions. Schematic representations ((A), (C), (F), and (H)), representative whole cell recordings ((B), (D), (G), (I)), and peak current density at +110 mV ((E) and (J)) are shown for each tested construct. HEK293 cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s in 20 mV increments. N=5-24 and error bars indicate SEM. Asterisks indicate significance of p<0.05 determined by one-way ANOVA followed by Dunnett’s test comparing the Panx1Δ356 (E) or Panx1 wild type (J) to each construct.

    Article Snippet: HEK293 cells (ATCC: CRL-1573) were cultured in DMEM supplemented with 10% FBS at 37°C with 8% CO2 in a humidified incubator.

    Techniques: Activation Assay, Construct

    Docked anions in the permeation pathway contribute to the channel closure. (A) Electrostatic free energy calculation of frPanx1-ΔC (red) in the presence of 1-4 ATP molecules in the deep energy well. (B) A docked ATP molecule on the NTD-flipped down human Panx1 (PDB: 7F8N). (C) Close-up view of the putative ATP binding pocket. Positive residues surrounding the docked ATP are indicated. (D) Peak current density at +110 mV of the wild type and alanine substitutions expressed in HEK293 cells. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s with 20 mV increments. N=4-15 and error bars indicate SEM. Asterisks indicate significance of p<0.05 determined by one-way ANOVA followed by Dunnett’s test comparing WT to each construct.

    Journal: bioRxiv

    Article Title: The C-terminal activating domain promotes Panx1 channel opening

    doi: 10.1101/2024.06.13.598903

    Figure Lengend Snippet: Docked anions in the permeation pathway contribute to the channel closure. (A) Electrostatic free energy calculation of frPanx1-ΔC (red) in the presence of 1-4 ATP molecules in the deep energy well. (B) A docked ATP molecule on the NTD-flipped down human Panx1 (PDB: 7F8N). (C) Close-up view of the putative ATP binding pocket. Positive residues surrounding the docked ATP are indicated. (D) Peak current density at +110 mV of the wild type and alanine substitutions expressed in HEK293 cells. Cells were clamped at -70 mV and stepped from -110 mV to +110 mV for 0.5 s with 20 mV increments. N=4-15 and error bars indicate SEM. Asterisks indicate significance of p<0.05 determined by one-way ANOVA followed by Dunnett’s test comparing WT to each construct.

    Article Snippet: HEK293 cells (ATCC: CRL-1573) were cultured in DMEM supplemented with 10% FBS at 37°C with 8% CO2 in a humidified incubator.

    Techniques: Binding Assay, Construct

    NaLTSA assay for SYK(WT)-NL with MRL-SYKi. ( A ) Melting curves with the addition of MRL-SYKi in dose–response format in HEK293 cells. Data reported are an average from biological triplicates each with two technical replicates. The data was fitted with a Boltzmann Sigmoid equation and error bars indicate the standard deviation ( B ) Visualization of the shift from vehicle with 30 μM of MRL-SYKi from ( A ). (C ) Melting temperatures (T m ) at each concentration of MRL-SYKi and the corresponding change in melting temperature (ΔT m ). Data are reported as an average of the T m ± SEM.

    Journal: bioRxiv

    Article Title: Development of SYK NanoBRET Cellular Target Engagement Assays for Gain–of–Function Variants

    doi: 10.1101/2024.06.12.598544

    Figure Lengend Snippet: NaLTSA assay for SYK(WT)-NL with MRL-SYKi. ( A ) Melting curves with the addition of MRL-SYKi in dose–response format in HEK293 cells. Data reported are an average from biological triplicates each with two technical replicates. The data was fitted with a Boltzmann Sigmoid equation and error bars indicate the standard deviation ( B ) Visualization of the shift from vehicle with 30 μM of MRL-SYKi from ( A ). (C ) Melting temperatures (T m ) at each concentration of MRL-SYKi and the corresponding change in melting temperature (ΔT m ). Data are reported as an average of the T m ± SEM.

    Article Snippet: Human embryonic kidney (HEK293) cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, #11965092) supplemented with 10% (v/v) fetal bovine serum (FBS, VWR Avantor Seradigm, #97068-085).

    Techniques: Standard Deviation, Concentration Assay

    SYK inhibitors bind potently to SYK GoF variants, reducing SYK phosphorylation and SYK catalytic activity. ( A ) IC 50 data for SYK inhibitors versus SYK GoF variants in HEK293 cells with NanoBRET tracer 8 . Data are reported as an average of triplicate IC 50 data ± SEM. Data reported are from a single experiment and plotted using a log(inhibitor) vs. response (three-parameter) fit. ( B ) Quantitative analysis of BRET using Cheng-Prusoff relationship for SYK variants in HEK293 cells with NanoBRET tracer 8 . The apparent K D value for MRL-SYKi was determined from the y-intercept by linear regression. Data reported are from a single experiment and plotted in GraphPad Prism with a simple linear regression. ( C ) The kinase activity of SYK(S550F) and SYK(P342T) is inhibited with MRL-SYKi in the PhosphoSens Assay. ( D ) MRL-SYKi decreases pSYK(Y525/526) levels in a SW480 cell line stably expressing SYK(S550Y) after treatment at 100 nM for 20 hrs.

    Journal: bioRxiv

    Article Title: Development of SYK NanoBRET Cellular Target Engagement Assays for Gain–of–Function Variants

    doi: 10.1101/2024.06.12.598544

    Figure Lengend Snippet: SYK inhibitors bind potently to SYK GoF variants, reducing SYK phosphorylation and SYK catalytic activity. ( A ) IC 50 data for SYK inhibitors versus SYK GoF variants in HEK293 cells with NanoBRET tracer 8 . Data are reported as an average of triplicate IC 50 data ± SEM. Data reported are from a single experiment and plotted using a log(inhibitor) vs. response (three-parameter) fit. ( B ) Quantitative analysis of BRET using Cheng-Prusoff relationship for SYK variants in HEK293 cells with NanoBRET tracer 8 . The apparent K D value for MRL-SYKi was determined from the y-intercept by linear regression. Data reported are from a single experiment and plotted in GraphPad Prism with a simple linear regression. ( C ) The kinase activity of SYK(S550F) and SYK(P342T) is inhibited with MRL-SYKi in the PhosphoSens Assay. ( D ) MRL-SYKi decreases pSYK(Y525/526) levels in a SW480 cell line stably expressing SYK(S550Y) after treatment at 100 nM for 20 hrs.

    Article Snippet: Human embryonic kidney (HEK293) cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, #11965092) supplemented with 10% (v/v) fetal bovine serum (FBS, VWR Avantor Seradigm, #97068-085).

    Techniques: Activity Assay, Stable Transfection, Expressing

    a , Arrangement of the lux genes in the expression cassette. b , In the bioluminescence reaction, light is generated through the concomitant oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain aliphatic aldehyde (RCHO), catalyzed by the luciferase (LuxAB). The two products are recycled by the fatty acid reductase complex (LuxCDE) and a flavin reductase (Frp). c , Comparison of luminescence emission from HEK-293 and HeLa cells transiently transfected with the expression cassette in a (1 plasmid) or a mixture of plasmids containing luxA, luxB, luxC, luxD, luxE and frp separately (6 plasmids). Plots show average values and standard deviations from 5 independent measurements.

    Journal: bioRxiv

    Article Title: Autonomous bioluminescence emission from transgenic mice

    doi: 10.1101/2024.06.13.598801

    Figure Lengend Snippet: a , Arrangement of the lux genes in the expression cassette. b , In the bioluminescence reaction, light is generated through the concomitant oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain aliphatic aldehyde (RCHO), catalyzed by the luciferase (LuxAB). The two products are recycled by the fatty acid reductase complex (LuxCDE) and a flavin reductase (Frp). c , Comparison of luminescence emission from HEK-293 and HeLa cells transiently transfected with the expression cassette in a (1 plasmid) or a mixture of plasmids containing luxA, luxB, luxC, luxD, luxE and frp separately (6 plasmids). Plots show average values and standard deviations from 5 independent measurements.

    Article Snippet: HeLa and HEK-293 cells were obtained from LGC Standards (cat. no. ATCC-CCL-2) and DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, cat. no. ACC 305), respectively.

    Techniques: Expressing, Generated, Luciferase, Comparison, Transfection, Plasmid Preparation