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    Figure 1. Interaction with <t>YES1</t> decreases the PM abundance and function of rF508del-CFTR in airway cells. (A) Intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR (r∆F, after rescue to the PM by 48 h treatment with 5 µM VX-661), were labelled with either an anti-Flag antibody or a non-specific IgG prior to non-denaturing lysis, and antibody-bound CFTR complexes were precipitated using protein G-coupled magnetic beads. Input protein levels were adjusted so that equivalent amounts of wt- and rF508del-CFTR were precipitated. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YES1. Tubulin (known to not interact with CFTR at the PM [24]) was used as an additional co-precipitation control. (B) Efficiency of siRNA-mediated depletion in CFBE cells expressing mCherry-F508del-CFTR, rescued as in (A), transfected with either a mock siRNA (siCtrl) or a commercial triple oligonucleotide mix against YES1 (siYES1). Representative WBs of CFTR, YES1, and tubulin (used as loading control) are shown (left panels), as well as the quantifications as means ± SEM from four independent experiments (right panel). (C) Immunofluorescence images of CFBE cells expressing Flag-tagged mCherry-F508del-CFTR rescued to the PM by 48 h treatment with 5 µM VX-661. The cells were transfected as in (B) and treated for 3 h with either vehicle (DMSO) or 10 µM of YES1 inhibitor SU6656. rF508del-CFTR at the surface of intact cells was immunolabelled on ice using an anti-Flag antibody followed by an Alexa 488-conjugated secondary antibody. Cells were then fixed, and the nuclei were stained with DAPI. Confocal images of the cells’ surface, showing surface CFTR staining in green (left panels) and a merged overlay image of surface (green) and total CFTR signals (mCherry, red) along with the stained nuclei (DAPI, blue) are shown in the right panels. White scale bars represent 10 µm. (D) Representative traces of ion transport activity measured through iodide-induced HS-YFP sensor fluorescence decay of untagged F508del-CFTR CFBE cells treated with 5 µM VX-661 for 48 h. The cells were transfected and treated as in (C) and co-treated with or without 25 µM of inh172 15 min prior to stimulation for 30 min in PBS with Fsk (5 µM) and Gen (10 µM), in the presence or absence of inh172. This was followed by continuous fluorescence recording and the addition of I- (represented by the black arrow, final concentration 100 mM). (E) Quantification of HS-YFP fluorescence quenching rates (QR) of at least five independent assays for each condition, calculated by fitting the iodide assay results to exponential decay curves. The means ± SEM are shown. ns—not significant, ** p < 0.01, and *** p < 0.001 between conditions indicated by the horizontal lines.
    Yes1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    yes1 - by Bioz Stars, 2026-09
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    1) Product Images from "YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1."

    Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

    Journal: Biomolecules

    doi: 10.3390/biom13060949

    Figure 1. Interaction with YES1 decreases the PM abundance and function of rF508del-CFTR in airway cells. (A) Intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR (r∆F, after rescue to the PM by 48 h treatment with 5 µM VX-661), were labelled with either an anti-Flag antibody or a non-specific IgG prior to non-denaturing lysis, and antibody-bound CFTR complexes were precipitated using protein G-coupled magnetic beads. Input protein levels were adjusted so that equivalent amounts of wt- and rF508del-CFTR were precipitated. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YES1. Tubulin (known to not interact with CFTR at the PM [24]) was used as an additional co-precipitation control. (B) Efficiency of siRNA-mediated depletion in CFBE cells expressing mCherry-F508del-CFTR, rescued as in (A), transfected with either a mock siRNA (siCtrl) or a commercial triple oligonucleotide mix against YES1 (siYES1). Representative WBs of CFTR, YES1, and tubulin (used as loading control) are shown (left panels), as well as the quantifications as means ± SEM from four independent experiments (right panel). (C) Immunofluorescence images of CFBE cells expressing Flag-tagged mCherry-F508del-CFTR rescued to the PM by 48 h treatment with 5 µM VX-661. The cells were transfected as in (B) and treated for 3 h with either vehicle (DMSO) or 10 µM of YES1 inhibitor SU6656. rF508del-CFTR at the surface of intact cells was immunolabelled on ice using an anti-Flag antibody followed by an Alexa 488-conjugated secondary antibody. Cells were then fixed, and the nuclei were stained with DAPI. Confocal images of the cells’ surface, showing surface CFTR staining in green (left panels) and a merged overlay image of surface (green) and total CFTR signals (mCherry, red) along with the stained nuclei (DAPI, blue) are shown in the right panels. White scale bars represent 10 µm. (D) Representative traces of ion transport activity measured through iodide-induced HS-YFP sensor fluorescence decay of untagged F508del-CFTR CFBE cells treated with 5 µM VX-661 for 48 h. The cells were transfected and treated as in (C) and co-treated with or without 25 µM of inh172 15 min prior to stimulation for 30 min in PBS with Fsk (5 µM) and Gen (10 µM), in the presence or absence of inh172. This was followed by continuous fluorescence recording and the addition of I- (represented by the black arrow, final concentration 100 mM). (E) Quantification of HS-YFP fluorescence quenching rates (QR) of at least five independent assays for each condition, calculated by fitting the iodide assay results to exponential decay curves. The means ± SEM are shown. ns—not significant, ** p < 0.01, and *** p < 0.001 between conditions indicated by the horizontal lines.
    Figure Legend Snippet: Figure 1. Interaction with YES1 decreases the PM abundance and function of rF508del-CFTR in airway cells. (A) Intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR (r∆F, after rescue to the PM by 48 h treatment with 5 µM VX-661), were labelled with either an anti-Flag antibody or a non-specific IgG prior to non-denaturing lysis, and antibody-bound CFTR complexes were precipitated using protein G-coupled magnetic beads. Input protein levels were adjusted so that equivalent amounts of wt- and rF508del-CFTR were precipitated. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YES1. Tubulin (known to not interact with CFTR at the PM [24]) was used as an additional co-precipitation control. (B) Efficiency of siRNA-mediated depletion in CFBE cells expressing mCherry-F508del-CFTR, rescued as in (A), transfected with either a mock siRNA (siCtrl) or a commercial triple oligonucleotide mix against YES1 (siYES1). Representative WBs of CFTR, YES1, and tubulin (used as loading control) are shown (left panels), as well as the quantifications as means ± SEM from four independent experiments (right panel). (C) Immunofluorescence images of CFBE cells expressing Flag-tagged mCherry-F508del-CFTR rescued to the PM by 48 h treatment with 5 µM VX-661. The cells were transfected as in (B) and treated for 3 h with either vehicle (DMSO) or 10 µM of YES1 inhibitor SU6656. rF508del-CFTR at the surface of intact cells was immunolabelled on ice using an anti-Flag antibody followed by an Alexa 488-conjugated secondary antibody. Cells were then fixed, and the nuclei were stained with DAPI. Confocal images of the cells’ surface, showing surface CFTR staining in green (left panels) and a merged overlay image of surface (green) and total CFTR signals (mCherry, red) along with the stained nuclei (DAPI, blue) are shown in the right panels. White scale bars represent 10 µm. (D) Representative traces of ion transport activity measured through iodide-induced HS-YFP sensor fluorescence decay of untagged F508del-CFTR CFBE cells treated with 5 µM VX-661 for 48 h. The cells were transfected and treated as in (C) and co-treated with or without 25 µM of inh172 15 min prior to stimulation for 30 min in PBS with Fsk (5 µM) and Gen (10 µM), in the presence or absence of inh172. This was followed by continuous fluorescence recording and the addition of I- (represented by the black arrow, final concentration 100 mM). (E) Quantification of HS-YFP fluorescence quenching rates (QR) of at least five independent assays for each condition, calculated by fitting the iodide assay results to exponential decay curves. The means ± SEM are shown. ns—not significant, ** p < 0.01, and *** p < 0.001 between conditions indicated by the horizontal lines.

    Techniques Used: Expressing, Lysis, Magnetic Beads, Control, Transfection, Staining, Activity Assay, Concentration Assay

    Figure 2. YES1 inhibition increases rF508del-CFTR retention at the PM upon thermal destabilization. (A) Diagram depicting the parallel cell surface protein biotinylation assays used to assess rF508del- CFTR thermal stability and internalization. Replicate dishes of CFBE cells expressing F508del-CFTR were incubated for 48 h at 30 ◦C in the presence of 5 µM of VX-661. One of the replicates was directly placed on ice, then the surface proteins were labeled with sulfo-NHS-SS-biotin, lysed, and the surface-labeled proteins were captured using streptavidin beads. These precipitates represented the input amount of CFTR at the PM without any thermal destabilization (DMSO 30 ◦C). A second set of replicates were moved to 37 ◦C in the absence (DMSO) or presence of YES1 inhibitors (10 µM SU6656 or 1 µM P505-15). Three hours later, surface proteins were labelled and isolated, as described above. This second set revealed the amount of CFTR remaining at the PM after thermal destabilization (TS) and inhibitor treatment. A third set of replicates was first labeled with biotin, then placed at 37 ◦C for 3 h in the presence or absence of YES inhibitors. These samples were then returned to ice, and the labeled CFTR remaining at the cell surface was stripped from biotin with glutathione prior to lysis and isolation of the labeled proteins that entered the cells. This third set represents the amount of CFTR internalized from the surface upon TS and inhibitor treatment. (B) Analysis of input lysates and biotin-labelled fractions obtained as described in (A). WBs representative of five independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut- 1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. (C) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in (B) after normalization to Glut-1 levels and to the respective controls. *** p < 0.001 relative to DMSO (30 ◦C), ## p < 0.01 and ### p < 0.001, both relative to DMSO in the internalized set.
    Figure Legend Snippet: Figure 2. YES1 inhibition increases rF508del-CFTR retention at the PM upon thermal destabilization. (A) Diagram depicting the parallel cell surface protein biotinylation assays used to assess rF508del- CFTR thermal stability and internalization. Replicate dishes of CFBE cells expressing F508del-CFTR were incubated for 48 h at 30 ◦C in the presence of 5 µM of VX-661. One of the replicates was directly placed on ice, then the surface proteins were labeled with sulfo-NHS-SS-biotin, lysed, and the surface-labeled proteins were captured using streptavidin beads. These precipitates represented the input amount of CFTR at the PM without any thermal destabilization (DMSO 30 ◦C). A second set of replicates were moved to 37 ◦C in the absence (DMSO) or presence of YES1 inhibitors (10 µM SU6656 or 1 µM P505-15). Three hours later, surface proteins were labelled and isolated, as described above. This second set revealed the amount of CFTR remaining at the PM after thermal destabilization (TS) and inhibitor treatment. A third set of replicates was first labeled with biotin, then placed at 37 ◦C for 3 h in the presence or absence of YES inhibitors. These samples were then returned to ice, and the labeled CFTR remaining at the cell surface was stripped from biotin with glutathione prior to lysis and isolation of the labeled proteins that entered the cells. This third set represents the amount of CFTR internalized from the surface upon TS and inhibitor treatment. (B) Analysis of input lysates and biotin-labelled fractions obtained as described in (A). WBs representative of five independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut- 1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. (C) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in (B) after normalization to Glut-1 levels and to the respective controls. *** p < 0.001 relative to DMSO (30 ◦C), ## p < 0.01 and ### p < 0.001, both relative to DMSO in the internalized set.

    Techniques Used: Inhibition, Expressing, Incubation, Labeling, Isolation, Lysis

    Figure 3. YAP1 is required for the binding of YES1 to rF508del-CFTR complexes at the PM. (A) STRING- based analysis (https://string-db.org/, accessed on 19 October 2021) of the strength of annotated evidence on the interaction between YES1 and the CFTR/NHERF1 (SLC9A3R1)/Ezrin (EZR) mem- brane anchoring complex (the thickness of the grey lines is proportional to the degree of confidence for the interaction between the two proteins they connect, extrapolated from text mining, experi- mental, and database-collected evidence). (B) A STRING-generated expanded interaction network, extended (green nodes) around the core complex in (A) (red nodes). (C) CFTR-containing complexes were immunoprecipitated from the PM of intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR, as described for Figure 1A. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YAP1. Tubulin was used as an additional co-precipitation control. (D) CFBE cells expressing untagged F508del-CFTR transfected with either mock siRNA (siCtrl) or a commercial siRNA mix targeting YAP1 (siYAP1), were incubated with 5 µM of VX-661 for 48 h at 30 ◦C to coax most of the mutant channel to the PM. The cells were then lysed in non-denaturing conditions and YES1 immunoprecipitated with a specific antibody (a non-specific IgG was used as a control) from whole cell lysates (WCL). Both input lysates and co-precipitates were analyzed using WB to assess the levels of precipitated YES1 and co-precipitated rF508del-CFTR. (E) Thermal shift (TS) assay, as described in Figure 2, to assess the thermal stability of untagged rF508del-CFTR in CFBE cells transfected with mock siRNA (siCtrl) or one of two commercial siRNA mixes targeting either YAP1 (siYAP1) or YES1 (siYES1). WBs repre- sentative of at least four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. (F) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in (E) after normalization to Glut-1 levels and to siCtrl (30 ◦C). *** p < 0.001 relative to siCtrl (30 ◦C), ## p < 0.01 and ### p < 0.001, both relative to siCtrl (TS).
    Figure Legend Snippet: Figure 3. YAP1 is required for the binding of YES1 to rF508del-CFTR complexes at the PM. (A) STRING- based analysis (https://string-db.org/, accessed on 19 October 2021) of the strength of annotated evidence on the interaction between YES1 and the CFTR/NHERF1 (SLC9A3R1)/Ezrin (EZR) mem- brane anchoring complex (the thickness of the grey lines is proportional to the degree of confidence for the interaction between the two proteins they connect, extrapolated from text mining, experi- mental, and database-collected evidence). (B) A STRING-generated expanded interaction network, extended (green nodes) around the core complex in (A) (red nodes). (C) CFTR-containing complexes were immunoprecipitated from the PM of intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR, as described for Figure 1A. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YAP1. Tubulin was used as an additional co-precipitation control. (D) CFBE cells expressing untagged F508del-CFTR transfected with either mock siRNA (siCtrl) or a commercial siRNA mix targeting YAP1 (siYAP1), were incubated with 5 µM of VX-661 for 48 h at 30 ◦C to coax most of the mutant channel to the PM. The cells were then lysed in non-denaturing conditions and YES1 immunoprecipitated with a specific antibody (a non-specific IgG was used as a control) from whole cell lysates (WCL). Both input lysates and co-precipitates were analyzed using WB to assess the levels of precipitated YES1 and co-precipitated rF508del-CFTR. (E) Thermal shift (TS) assay, as described in Figure 2, to assess the thermal stability of untagged rF508del-CFTR in CFBE cells transfected with mock siRNA (siCtrl) or one of two commercial siRNA mixes targeting either YAP1 (siYAP1) or YES1 (siYES1). WBs repre- sentative of at least four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. (F) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in (E) after normalization to Glut-1 levels and to siCtrl (30 ◦C). *** p < 0.001 relative to siCtrl (30 ◦C), ## p < 0.01 and ### p < 0.001, both relative to siCtrl (TS).

    Techniques Used: Binding Assay, Generated, Immunoprecipitation, Expressing, Control, Transfection, Incubation, Mutagenesis

    Figure 4. The MAPK pathway participates in YES1-mediated internalization of rF508del-CFTR at the PM. The thermal shift (TS) assay, as described in Figure 2, was used to assess the thermal stability of untagged rF508del-CFTR in CFBE cells. (A) The cells were treated for 3 h with either vehicle (DMSO) or 10 µM of selumetinib, or (C) transfected with empty vector or Myc-H-RAS-V12 (HRAS) and then treated for 3 h with vehicle (DMSO) or 10 µM of SU6656, as indicated. WBs representative of input and cell surface fractions from four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. H-RAS V12 was detected using an anti-Myc antibody, and an anti-phosphorylated ERK1/2 antibody was used to monitor MAPK path- way activity, which was quantified and shown below the respective blot lanes. (B,D) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from four independent assays after normalization to Glut-1 levels and DMSO (30 ◦C). * p < 0.05, ** p < 0.01, and *** p < 0.001, relative to DMSO (30 ◦C) in (B), and as indicated by the horizontal lines in (C); ## p < 0.01, relative to DMSO (TS) in (B).
    Figure Legend Snippet: Figure 4. The MAPK pathway participates in YES1-mediated internalization of rF508del-CFTR at the PM. The thermal shift (TS) assay, as described in Figure 2, was used to assess the thermal stability of untagged rF508del-CFTR in CFBE cells. (A) The cells were treated for 3 h with either vehicle (DMSO) or 10 µM of selumetinib, or (C) transfected with empty vector or Myc-H-RAS-V12 (HRAS) and then treated for 3 h with vehicle (DMSO) or 10 µM of SU6656, as indicated. WBs representative of input and cell surface fractions from four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. H-RAS V12 was detected using an anti-Myc antibody, and an anti-phosphorylated ERK1/2 antibody was used to monitor MAPK path- way activity, which was quantified and shown below the respective blot lanes. (B,D) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from four independent assays after normalization to Glut-1 levels and DMSO (30 ◦C). * p < 0.05, ** p < 0.01, and *** p < 0.001, relative to DMSO (30 ◦C) in (B), and as indicated by the horizontal lines in (C); ## p < 0.01, relative to DMSO (TS) in (B).

    Techniques Used: Transfection, Plasmid Preparation, Activity Assay

    Figure 5. SHC1 phosphorylation by YES1 mediates rF508del-CFTR internalization via MAPK path- way signaling. (A) Effects of YES1 inhibitors. Lysates from F508del-CFTR expressing CFBE cells were incubated with 5 µM of VX-661 for 48 h at 30 ◦C, treated with either vehicle (DMSO), SU6656 (10 µM), or P505-15 (1 µM) for 3 h at 37 ◦C, then were analyzed using WB. Immunoblots representa- tive of three independent experiments, probed with antibodies against the indicated proteins, are shown. p-SHC1 indicates the level of SHC1 phosphorylation at Tyr239/240 in the different conditions, and an anti-phosphorylated ERK1/2 antibody (p-ERK1/2) was used to monitor MAPK pathway activity (both show quantified band intensities below their respective blots). (B) Thermal shift (TS) assay described in Figure 2 to assess how much rF508del-CFTR remained at the PM after thermal destabilization in CFBE cells transfected either with a mock siRNA (siCtrl) or a siRNA against SHC1 (siSHC1). WBs representative of input and cell surface fractions, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. Quantifications of SCH1 depletion efficiency and ERK1/2 phosphorylation levels are shown below their respective blots. (C) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from three independent assays after normalization to Glut-1 levels and to siCtrl (30 ◦C). *** p < 0.001 relative to siCtrl (30 ◦C), ## p < 0.01 relative to siCtrl (TS).
    Figure Legend Snippet: Figure 5. SHC1 phosphorylation by YES1 mediates rF508del-CFTR internalization via MAPK path- way signaling. (A) Effects of YES1 inhibitors. Lysates from F508del-CFTR expressing CFBE cells were incubated with 5 µM of VX-661 for 48 h at 30 ◦C, treated with either vehicle (DMSO), SU6656 (10 µM), or P505-15 (1 µM) for 3 h at 37 ◦C, then were analyzed using WB. Immunoblots representa- tive of three independent experiments, probed with antibodies against the indicated proteins, are shown. p-SHC1 indicates the level of SHC1 phosphorylation at Tyr239/240 in the different conditions, and an anti-phosphorylated ERK1/2 antibody (p-ERK1/2) was used to monitor MAPK pathway activity (both show quantified band intensities below their respective blots). (B) Thermal shift (TS) assay described in Figure 2 to assess how much rF508del-CFTR remained at the PM after thermal destabilization in CFBE cells transfected either with a mock siRNA (siCtrl) or a siRNA against SHC1 (siSHC1). WBs representative of input and cell surface fractions, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. Quantifications of SCH1 depletion efficiency and ERK1/2 phosphorylation levels are shown below their respective blots. (C) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from three independent assays after normalization to Glut-1 levels and to siCtrl (30 ◦C). *** p < 0.001 relative to siCtrl (30 ◦C), ## p < 0.01 relative to siCtrl (TS).

    Techniques Used: Phospho-proteomics, Expressing, Incubation, Western Blot, Activity Assay, Transfection

    Figure 6. Proposed model for SHC1-mediated removal of CFTR from the PM through activation of the MAPK pathway. (A) Phosphorylation of PM-anchored wt-CFTR at Tyr512 by SYK kinase leads to its recognition and binding by the adaptor protein SHC1. This links CFTR internalization to the activation of the MAPK pathway downstream of receptor tyrosine kinases. (B) F508del-CFTR pharmacologically rescued to the PM is not phosphorylated by SYK, but its deficient anchoring to the actin cytoskeleton allows its interaction with the YES1 kinase via the adaptor protein YAP1 and the scaffold protein NHERF1. SHC1 is a substrate for YES1 at the PM, and its phosphorylation by YES1 increases its affinity to membrane receptors in the vicinity, enhancing their activation of the MAPK pathway. This could contribute to the much faster internalization rate of rF508del-CFTR compared to the wild-type protein. RTK—receptor tyrosine kinases; EB—Ezrin binding domain; 1-2—NHERF1’s PDZ1 and PDZ2.
    Figure Legend Snippet: Figure 6. Proposed model for SHC1-mediated removal of CFTR from the PM through activation of the MAPK pathway. (A) Phosphorylation of PM-anchored wt-CFTR at Tyr512 by SYK kinase leads to its recognition and binding by the adaptor protein SHC1. This links CFTR internalization to the activation of the MAPK pathway downstream of receptor tyrosine kinases. (B) F508del-CFTR pharmacologically rescued to the PM is not phosphorylated by SYK, but its deficient anchoring to the actin cytoskeleton allows its interaction with the YES1 kinase via the adaptor protein YAP1 and the scaffold protein NHERF1. SHC1 is a substrate for YES1 at the PM, and its phosphorylation by YES1 increases its affinity to membrane receptors in the vicinity, enhancing their activation of the MAPK pathway. This could contribute to the much faster internalization rate of rF508del-CFTR compared to the wild-type protein. RTK—receptor tyrosine kinases; EB—Ezrin binding domain; 1-2—NHERF1’s PDZ1 and PDZ2.

    Techniques Used: Activation Assay, Phospho-proteomics, Binding Assay, Membrane

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    Santa Cruz Biotechnology yes1
    Figure 1. Interaction with <t>YES1</t> decreases the PM abundance and function of rF508del-CFTR in airway cells. (A) Intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR (r∆F, after rescue to the PM by 48 h treatment with 5 µM VX-661), were labelled with either an anti-Flag antibody or a non-specific IgG prior to non-denaturing lysis, and antibody-bound CFTR complexes were precipitated using protein G-coupled magnetic beads. Input protein levels were adjusted so that equivalent amounts of wt- and rF508del-CFTR were precipitated. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YES1. Tubulin (known to not interact with CFTR at the PM [24]) was used as an additional co-precipitation control. (B) Efficiency of siRNA-mediated depletion in CFBE cells expressing mCherry-F508del-CFTR, rescued as in (A), transfected with either a mock siRNA (siCtrl) or a commercial triple oligonucleotide mix against YES1 (siYES1). Representative WBs of CFTR, YES1, and tubulin (used as loading control) are shown (left panels), as well as the quantifications as means ± SEM from four independent experiments (right panel). (C) Immunofluorescence images of CFBE cells expressing Flag-tagged mCherry-F508del-CFTR rescued to the PM by 48 h treatment with 5 µM VX-661. The cells were transfected as in (B) and treated for 3 h with either vehicle (DMSO) or 10 µM of YES1 inhibitor SU6656. rF508del-CFTR at the surface of intact cells was immunolabelled on ice using an anti-Flag antibody followed by an Alexa 488-conjugated secondary antibody. Cells were then fixed, and the nuclei were stained with DAPI. Confocal images of the cells’ surface, showing surface CFTR staining in green (left panels) and a merged overlay image of surface (green) and total CFTR signals (mCherry, red) along with the stained nuclei (DAPI, blue) are shown in the right panels. White scale bars represent 10 µm. (D) Representative traces of ion transport activity measured through iodide-induced HS-YFP sensor fluorescence decay of untagged F508del-CFTR CFBE cells treated with 5 µM VX-661 for 48 h. The cells were transfected and treated as in (C) and co-treated with or without 25 µM of inh172 15 min prior to stimulation for 30 min in PBS with Fsk (5 µM) and Gen (10 µM), in the presence or absence of inh172. This was followed by continuous fluorescence recording and the addition of I- (represented by the black arrow, final concentration 100 mM). (E) Quantification of HS-YFP fluorescence quenching rates (QR) of at least five independent assays for each condition, calculated by fitting the iodide assay results to exponential decay curves. The means ± SEM are shown. ns—not significant, ** p < 0.01, and *** p < 0.001 between conditions indicated by the horizontal lines.
    Yes1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/yes+sirna/c-Yes+siRNA/pm37371529-52-22-39
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    yes1 sirna pool of 3 different sirna duplexes  (Santa Cruz Biotechnology)
    90
    Santa Cruz Biotechnology yes1 sirna pool of 3 different sirna duplexes
    Figure 1. Interaction with <t>YES1</t> decreases the PM abundance and function of rF508del-CFTR in airway cells. (A) Intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR (r∆F, after rescue to the PM by 48 h treatment with 5 µM VX-661), were labelled with either an anti-Flag antibody or a non-specific IgG prior to non-denaturing lysis, and antibody-bound CFTR complexes were precipitated using protein G-coupled magnetic beads. Input protein levels were adjusted so that equivalent amounts of wt- and rF508del-CFTR were precipitated. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YES1. Tubulin (known to not interact with CFTR at the PM [24]) was used as an additional co-precipitation control. (B) Efficiency of siRNA-mediated depletion in CFBE cells expressing mCherry-F508del-CFTR, rescued as in (A), transfected with either a mock siRNA (siCtrl) or a commercial triple oligonucleotide mix against YES1 (siYES1). Representative WBs of CFTR, YES1, and tubulin (used as loading control) are shown (left panels), as well as the quantifications as means ± SEM from four independent experiments (right panel). (C) Immunofluorescence images of CFBE cells expressing Flag-tagged mCherry-F508del-CFTR rescued to the PM by 48 h treatment with 5 µM VX-661. The cells were transfected as in (B) and treated for 3 h with either vehicle (DMSO) or 10 µM of YES1 inhibitor SU6656. rF508del-CFTR at the surface of intact cells was immunolabelled on ice using an anti-Flag antibody followed by an Alexa 488-conjugated secondary antibody. Cells were then fixed, and the nuclei were stained with DAPI. Confocal images of the cells’ surface, showing surface CFTR staining in green (left panels) and a merged overlay image of surface (green) and total CFTR signals (mCherry, red) along with the stained nuclei (DAPI, blue) are shown in the right panels. White scale bars represent 10 µm. (D) Representative traces of ion transport activity measured through iodide-induced HS-YFP sensor fluorescence decay of untagged F508del-CFTR CFBE cells treated with 5 µM VX-661 for 48 h. The cells were transfected and treated as in (C) and co-treated with or without 25 µM of inh172 15 min prior to stimulation for 30 min in PBS with Fsk (5 µM) and Gen (10 µM), in the presence or absence of inh172. This was followed by continuous fluorescence recording and the addition of I- (represented by the black arrow, final concentration 100 mM). (E) Quantification of HS-YFP fluorescence quenching rates (QR) of at least five independent assays for each condition, calculated by fitting the iodide assay results to exponential decay curves. The means ± SEM are shown. ns—not significant, ** p < 0.01, and *** p < 0.001 between conditions indicated by the horizontal lines.
    Yes1 Sirna Pool Of 3 Different Sirna Duplexes, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    small interfering rna (sirna) yes-associated protein ( yap)  (Ribobio co)
    90
    Ribobio co small interfering rna (sirna) yes-associated protein ( yap)
    Effects of <t>cGAS/STING</t> knockdown on the migration and invasion of RA FLSs induced by cytosolic dsDNA. RA FLSs were transfected with <t>siRNA</t> or ISD dsDNA (2 µg/mL). (A) RA FLSs were transfected with cGAS or STING siRNA (sicGAS1-3 or siSTING1-3) or control siRNA (siC) for 48 hours and subjected to qRT-PCR analysis for mRNA expression or western blot analysis for protein expression. A representative blot of at least 3 independent experiments is shown. (B,C) Migration and invasion of RA FLSs were measured using a Transwell assay. Invasion was evaluated using inserts coated with matrigel basement membrane matrix. The relative migratory or invasive rate was calculated by counting migrated or invaded cells and then normalized to the siC or siC + dsDNA. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. The graphs show the relative migration rates. (D) cGAS or STING knockdown impaired the lamellipodia formation in RA FLSs. RA FLSs were wounded and stimulated with dsDNA (2 µg/mL) for 8 hours. Representative images are shown (original magnification: 400×). The yellow arrow indicates lamellipodia formation. The data (A-C) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. siC; #P<0.05; ##P<0.01 vs. siC + dsDNA. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; sicGAS, cGAS siRNA; siSTING, STING siRNA; siC, control siRNA; dsDNA, double-stranded DNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.
    Small Interfering Rna (Sirna) Yes Associated Protein ( Yap), supplied by Ribobio co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    3 sirna targeting yes1  (Shanghai GenePharma)
    90
    Shanghai GenePharma 3 sirna targeting yes1
    A Venn diagram depicting 9 candidate common targets of miR-145-5p and linc01133. B The complementary sequence of <t>YES1</t> 3’UTR with miR-145-5p seed sequence predicted by TargetScan and the mutated sequence. MiR-145-5p reduced the luciferase activity of pmirGLO-YES1 3’UTR but not that with miR-145-5p binding site mutation in MKN45 ( C ) and HEK293 cells ( D ). MiR-145-5p inhibited YES1 expression in both mRNA ( E )and protein level ( F ). G YES1 3’UTR is AGO2 bounded demonstrated by RIP assay. H YES1 could be pulled down by biotin-labeled miR-145-5p. Linc01133 overexpression in HGC27 cells or knocking-down in AGS cells increased or decreased the expression of YES1 both in mRNA ( I, J ) and protein level ( K ). L Linc01133 greatly attenuated miR-145-5p induced YES1 down-regulation. Each experiment was performed in triplicates. Data are shown as mean ± SD, * P < 0.05, ** P < 0.01.
    3 Sirna Targeting Yes1, supplied by Shanghai GenePharma, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    yes-associated protein sirna yap-sirna  (Shanghai GenePharma)
    90
    Shanghai GenePharma yes-associated protein sirna yap-sirna
    A Venn diagram depicting 9 candidate common targets of miR-145-5p and linc01133. B The complementary sequence of <t>YES1</t> 3’UTR with miR-145-5p seed sequence predicted by TargetScan and the mutated sequence. MiR-145-5p reduced the luciferase activity of pmirGLO-YES1 3’UTR but not that with miR-145-5p binding site mutation in MKN45 ( C ) and HEK293 cells ( D ). MiR-145-5p inhibited YES1 expression in both mRNA ( E )and protein level ( F ). G YES1 3’UTR is AGO2 bounded demonstrated by RIP assay. H YES1 could be pulled down by biotin-labeled miR-145-5p. Linc01133 overexpression in HGC27 cells or knocking-down in AGS cells increased or decreased the expression of YES1 both in mRNA ( I, J ) and protein level ( K ). L Linc01133 greatly attenuated miR-145-5p induced YES1 down-regulation. Each experiment was performed in triplicates. Data are shown as mean ± SD, * P < 0.05, ** P < 0.01.
    Yes Associated Protein Sirna Yap Sirna, supplied by Shanghai GenePharma, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/yes+sirna/yes+associated+protein+sirna+yap+sirna/10__1016_slash_j__jmst__2020__03__009-57-8-19
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    sirna specific for yes1 silencer1 select validated sirna #4390824  (Thermo Fisher)
    90
    Thermo Fisher sirna specific for yes1 silencer1 select validated sirna #4390824
    A Venn diagram depicting 9 candidate common targets of miR-145-5p and linc01133. B The complementary sequence of <t>YES1</t> 3’UTR with miR-145-5p seed sequence predicted by TargetScan and the mutated sequence. MiR-145-5p reduced the luciferase activity of pmirGLO-YES1 3’UTR but not that with miR-145-5p binding site mutation in MKN45 ( C ) and HEK293 cells ( D ). MiR-145-5p inhibited YES1 expression in both mRNA ( E )and protein level ( F ). G YES1 3’UTR is AGO2 bounded demonstrated by RIP assay. H YES1 could be pulled down by biotin-labeled miR-145-5p. Linc01133 overexpression in HGC27 cells or knocking-down in AGS cells increased or decreased the expression of YES1 both in mRNA ( I, J ) and protein level ( K ). L Linc01133 greatly attenuated miR-145-5p induced YES1 down-regulation. Each experiment was performed in triplicates. Data are shown as mean ± SD, * P < 0.05, ** P < 0.01.
    Sirna Specific For Yes1 Silencer1 Select Validated Sirna #4390824, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/yes+sirna/silencer1+sirnas/pm31856375-56-0-24
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    Image Search Results


    (A) Heatmap showing the effects of Cul9 levels on the transcriptomics in GC TCGA database. (B) The enrichment of top 20 function pathways modulated by Cul9 expression in GC patients. (C) Cul9 was knocked down in BGC823 and HGC27 cell lines. Mass spectrometry was performed. The Venn diagram indicated the overlap of proteins differentially expressed in BGC823 and HGC27 cells transfected with shCul9-1 # or 2 #. And the heatmap of the upregulated and downregulated proteins was generated to predict potential Cul9-associated tyrosine kinase candidates. Finally, Yes1 was selected according to the overlaps. (D) BGC823 and HGC27 cells with Cul9 knockdown were treated with or without 10 mmol/l 416 kinase inhibitors, and cell viability was analyzed. The relative effects of these inhibitors were presented using the heat map. The top three inhibitors having anti-GC activity were listed. (Ei–iv) BGC823 and HGC27 cells with Cul9 knockdown were treated with or without 10 mmol/l Yes1 inhibitor CH6953755. Indicated experiments were performed (n=3, *P < 0.05). (F) BGC823 and HGC27 cells with Cul9 knockdown were transfected with Yes1 siRNA for 48 hours. Then examined the cell viability. Data are shown as mean ± SEM; **P < 0.01; ***P < 0.001.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: (A) Heatmap showing the effects of Cul9 levels on the transcriptomics in GC TCGA database. (B) The enrichment of top 20 function pathways modulated by Cul9 expression in GC patients. (C) Cul9 was knocked down in BGC823 and HGC27 cell lines. Mass spectrometry was performed. The Venn diagram indicated the overlap of proteins differentially expressed in BGC823 and HGC27 cells transfected with shCul9-1 # or 2 #. And the heatmap of the upregulated and downregulated proteins was generated to predict potential Cul9-associated tyrosine kinase candidates. Finally, Yes1 was selected according to the overlaps. (D) BGC823 and HGC27 cells with Cul9 knockdown were treated with or without 10 mmol/l 416 kinase inhibitors, and cell viability was analyzed. The relative effects of these inhibitors were presented using the heat map. The top three inhibitors having anti-GC activity were listed. (Ei–iv) BGC823 and HGC27 cells with Cul9 knockdown were treated with or without 10 mmol/l Yes1 inhibitor CH6953755. Indicated experiments were performed (n=3, *P < 0.05). (F) BGC823 and HGC27 cells with Cul9 knockdown were transfected with Yes1 siRNA for 48 hours. Then examined the cell viability. Data are shown as mean ± SEM; **P < 0.01; ***P < 0.001.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Expressing, Mass Spectrometry, Transfection, Generated, Activity Assay

    (A) The protein levels of Yes1 were examined in Cul9 +/+ and Cul9 −/− MEFs. (B) Endogenous Yes1 levels were determined in GC cells transfected with Flag-Cul9 by western analysis using indicated antibodies. (C) Cul9 was knocked down in SGC7901 cells with two siRNAs. The protein and mRNA levels of Yes1 were examined by Western blots and RT-qPCR, respectively. (D) The cycloheximide-chase experiments were carried out to show the half-life of Yes1 protein in Cul9 +/+ and cul9 −/− MEFs. (E) Yes1 ubiquitination in vivo was analyzed using the immunoprecipitates from HGC27 cells which were transfected with scramble or siCul9 , HA-ubiquitin, Flag–Yes1 , and then treated with H.pylori or MG132 together. (F) In vitro ubiquitination assay. The immunoprecipitated Cul9, recombinant Yes1, WT, or methylated (Met) ubiquitin (a negative control). (G) MS/MS identified K468 as a Yes1 ubiquitination site by Cul9, and K468 was conserved residue in Yes1. (H) In vitro ubiquitination assay. The immunoprecipitated Cul9 combined with WT-Yes1, or K468R mutant, and recombinant HA-ubiquitin were used. (I) CCK8 assays indicated that the K468R mutant of Yes1 promoted SGC7901 cell proliferation compared with Yes1-WT. Data are shown as mean ± SEM; **P < 0.01.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: (A) The protein levels of Yes1 were examined in Cul9 +/+ and Cul9 −/− MEFs. (B) Endogenous Yes1 levels were determined in GC cells transfected with Flag-Cul9 by western analysis using indicated antibodies. (C) Cul9 was knocked down in SGC7901 cells with two siRNAs. The protein and mRNA levels of Yes1 were examined by Western blots and RT-qPCR, respectively. (D) The cycloheximide-chase experiments were carried out to show the half-life of Yes1 protein in Cul9 +/+ and cul9 −/− MEFs. (E) Yes1 ubiquitination in vivo was analyzed using the immunoprecipitates from HGC27 cells which were transfected with scramble or siCul9 , HA-ubiquitin, Flag–Yes1 , and then treated with H.pylori or MG132 together. (F) In vitro ubiquitination assay. The immunoprecipitated Cul9, recombinant Yes1, WT, or methylated (Met) ubiquitin (a negative control). (G) MS/MS identified K468 as a Yes1 ubiquitination site by Cul9, and K468 was conserved residue in Yes1. (H) In vitro ubiquitination assay. The immunoprecipitated Cul9 combined with WT-Yes1, or K468R mutant, and recombinant HA-ubiquitin were used. (I) CCK8 assays indicated that the K468R mutant of Yes1 promoted SGC7901 cell proliferation compared with Yes1-WT. Data are shown as mean ± SEM; **P < 0.01.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Transfection, Western Blot, Quantitative RT-PCR, In Vivo, In Vitro, Ubiquitin Assay, Immunoprecipitation, Recombinant, Methylation, Negative Control, Tandem Mass Spectroscopy, Residue, Mutagenesis

    (A) The proximity-dependent biotin (BioID2) experiment identified Yes1 in Cul9-complex. (Bi) HEK293T cells were co-transfected with HA-Cul9 and Flag-Yes1 for 48 hours. The indicated immunoprecipitation and subsequent immunoblotting were performed with HA or Flag antibodies. (Bii) GC cells were immunoprecipitated with an anti-Yes1 or anti-Cul9 antibody and probed with indicated antibodies. (Ci) Binding of different human Yes1 truncated fragments to Cul9 as indicated (left panel). (Cii) Binding assays about the interaction of Cul9 domains with Yes1 (right panel). The immunoblots and Coomassie blue staining were performed. (D) Proximity ligation assay indicated that H.pylori transfection promoted the binding of Cul9 to Yes1 in the cytoplasm of SGC7901 cells. (E) In vitro kinase analysis using inactive or active Yes1, ATP, and GST-Cul9. 4G10 antibody detected Cul9 tyrosine phosphorylation. (F) Cell lysates were prepared from SGC7901 cells with Yes1 silencing by using siRNA. Phosphorylation of Cul9 was evaluated by using the indicated antibodies. (Gi and Giii) MS/MS identified Y1505 as a majorly phosphorylated site of Cul9, which is localized in the conserved phosphorylation motif of Yes1. (Gii) In vitro Kinase assays were performed with purified GST-Cul9 and its mutant Y1505F. Phosphorylation of Cul9 was evaluated by Western blot with an 4G10 antibody. (H) GC cells were treated with or without H. pylori for 6 hours and subjected to immunoblotting analyses with a specific Cul9-Y1505 phosphorylation antibody.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: (A) The proximity-dependent biotin (BioID2) experiment identified Yes1 in Cul9-complex. (Bi) HEK293T cells were co-transfected with HA-Cul9 and Flag-Yes1 for 48 hours. The indicated immunoprecipitation and subsequent immunoblotting were performed with HA or Flag antibodies. (Bii) GC cells were immunoprecipitated with an anti-Yes1 or anti-Cul9 antibody and probed with indicated antibodies. (Ci) Binding of different human Yes1 truncated fragments to Cul9 as indicated (left panel). (Cii) Binding assays about the interaction of Cul9 domains with Yes1 (right panel). The immunoblots and Coomassie blue staining were performed. (D) Proximity ligation assay indicated that H.pylori transfection promoted the binding of Cul9 to Yes1 in the cytoplasm of SGC7901 cells. (E) In vitro kinase analysis using inactive or active Yes1, ATP, and GST-Cul9. 4G10 antibody detected Cul9 tyrosine phosphorylation. (F) Cell lysates were prepared from SGC7901 cells with Yes1 silencing by using siRNA. Phosphorylation of Cul9 was evaluated by using the indicated antibodies. (Gi and Giii) MS/MS identified Y1505 as a majorly phosphorylated site of Cul9, which is localized in the conserved phosphorylation motif of Yes1. (Gii) In vitro Kinase assays were performed with purified GST-Cul9 and its mutant Y1505F. Phosphorylation of Cul9 was evaluated by Western blot with an 4G10 antibody. (H) GC cells were treated with or without H. pylori for 6 hours and subjected to immunoblotting analyses with a specific Cul9-Y1505 phosphorylation antibody.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, Staining, Proximity Ligation Assay, In Vitro, Tandem Mass Spectroscopy, Purification, Mutagenesis

    (A) Flag-Yes1 was transfected into HEK293T cells for 48 hours with or without His-Cul9 . Western blots were performed as described. (B) HEK293T cells were transfected with Flag-Cul9 and WT-His-Yes1 or Yes1-KD mutant. The levels of Cul9 mRNA were examined. (C) [ 35 S]-methionine was used to label the cells with SGC7901- shcontrol and SGC7901- shYes1 , and chasing with a standard medium was followed. (D) The endogenous Cul9 expression was examined in (C). (E) The CHX assays were performed. (F) After Flag-Yes1 and His-Cul9 co-transfection, HEK293T cells were treated using NH 4 Cl (20mM), 3MA (10mM), MG132 (10μM), CQ (50μM), or BafA1 (0.2μM). Then immunoblots were performed. (Gi and ii) The effects of Beclin1 or ATG5 knockdown in Cul9 -transfected HEK293T cells. (H) BafA1 increases Cul9 levels in SGC7901 cells for the duration indicated.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: (A) Flag-Yes1 was transfected into HEK293T cells for 48 hours with or without His-Cul9 . Western blots were performed as described. (B) HEK293T cells were transfected with Flag-Cul9 and WT-His-Yes1 or Yes1-KD mutant. The levels of Cul9 mRNA were examined. (C) [ 35 S]-methionine was used to label the cells with SGC7901- shcontrol and SGC7901- shYes1 , and chasing with a standard medium was followed. (D) The endogenous Cul9 expression was examined in (C). (E) The CHX assays were performed. (F) After Flag-Yes1 and His-Cul9 co-transfection, HEK293T cells were treated using NH 4 Cl (20mM), 3MA (10mM), MG132 (10μM), CQ (50μM), or BafA1 (0.2μM). Then immunoblots were performed. (Gi and ii) The effects of Beclin1 or ATG5 knockdown in Cul9 -transfected HEK293T cells. (H) BafA1 increases Cul9 levels in SGC7901 cells for the duration indicated.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Transfection, Western Blot, Mutagenesis, Expressing, Cotransfection

    (A) After Yes1 , p62 , and OPTN transfection , HEK293T cells were immunoprecipitated with anti-Cul9. Then performing immunoblot analysis as indicated. (B) Yes1 knockdown disrupted the binding of Cul9 to p62 in SGC7901 cells. (C) HEK293T cells were transfected as indicated, and co-immunoprecipitation was performed. (Di-ii) HEK293T cells with p62 or OPTN knockdown or control cells were transfected with Flag-Yes1 . The immunoblots were performed. (E) Control or p62 -knockdown HEK293T cells were treated with 100μg/mL CHX as indicated. Then examine the levels of Cul9 protein. (F) Y1505F mutation reduced Cul9-K63 ubiquitination. (G) 15 mM NH 4 Cl treatment increased K63-poly-ubiquitination of Cul9. (H) Vanadate addition enhanced Cul9 ubiquitination.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: (A) After Yes1 , p62 , and OPTN transfection , HEK293T cells were immunoprecipitated with anti-Cul9. Then performing immunoblot analysis as indicated. (B) Yes1 knockdown disrupted the binding of Cul9 to p62 in SGC7901 cells. (C) HEK293T cells were transfected as indicated, and co-immunoprecipitation was performed. (Di-ii) HEK293T cells with p62 or OPTN knockdown or control cells were transfected with Flag-Yes1 . The immunoblots were performed. (E) Control or p62 -knockdown HEK293T cells were treated with 100μg/mL CHX as indicated. Then examine the levels of Cul9 protein. (F) Y1505F mutation reduced Cul9-K63 ubiquitination. (G) 15 mM NH 4 Cl treatment increased K63-poly-ubiquitination of Cul9. (H) Vanadate addition enhanced Cul9 ubiquitination.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, Mutagenesis

    HEK293T cells were infected using the indicated constructs (A ) or scramble or siCOP1 (B). Then the co-IP and western blots were carried out as indicated. (Ci) The conserved VP motif in Cul9. (Cii) Co-IPs were performed in HEK293T cells as indicated. (Di) The immunoblots were performed in GC cells as indicated. (Dii) The immunoblots were performed in GC cells with Yes1 knockdown or not as indicated. (E) MS/MS identified Cul9 ubiquitination residues. (F) GC cells were used for in vivo ubiquitination experiments. (G) The effects of WT-Cul9 and its K1657R mutant in GC cells.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: HEK293T cells were infected using the indicated constructs (A ) or scramble or siCOP1 (B). Then the co-IP and western blots were carried out as indicated. (Ci) The conserved VP motif in Cul9. (Cii) Co-IPs were performed in HEK293T cells as indicated. (Di) The immunoblots were performed in GC cells as indicated. (Dii) The immunoblots were performed in GC cells with Yes1 knockdown or not as indicated. (E) MS/MS identified Cul9 ubiquitination residues. (F) GC cells were used for in vivo ubiquitination experiments. (G) The effects of WT-Cul9 and its K1657R mutant in GC cells.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Infection, Construct, Co-Immunoprecipitation Assay, Western Blot, Tandem Mass Spectroscopy, In Vivo, Mutagenesis

    (A) The levels of Yes1 in SGC7901 cells were challenged with or without CagA+ HP strains 7.13 or J166. MOI means a multiplicity of infections. (Bi) Western blot assays for the samples from (A) treated with BafA1. (Bii) SGC7901 cells infected with 7.13 and J166. The experiments were performed as indicated. (C) Yes1 activity was analyzed. (D) Mice were challenged with HP strain PMSS1 or Brucella broth as a control. QRT-PCR (Di) and Western blots (Dii-iii) were performed. (Ei) Yes1 architecture domain and its GC-related mutants in GC patients. (Eii) Relative activity of Yes1 was analyzed. (Eiii) IP kinase assay. Briefly, Yes1 was immunoprecipitated from HEK293 cells expressing Flag-tagged WT-Yes1 or its mutants. Then the immunoprecipitated Yes1 was mixed with [ 32 P] ATP and a known substrate peptide of Yes1. The results were normalized to 1.0 for WT-Yes1. (Fi-ii) The effects of GC-related Yes1 mutants on GC cells. (Gi-ii) The invasion and migration of GC cells were affected by GC-associated Yes1 mutants. Data are shown as mean ± SEM; **P < 0.01; ***P < 0.001.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: (A) The levels of Yes1 in SGC7901 cells were challenged with or without CagA+ HP strains 7.13 or J166. MOI means a multiplicity of infections. (Bi) Western blot assays for the samples from (A) treated with BafA1. (Bii) SGC7901 cells infected with 7.13 and J166. The experiments were performed as indicated. (C) Yes1 activity was analyzed. (D) Mice were challenged with HP strain PMSS1 or Brucella broth as a control. QRT-PCR (Di) and Western blots (Dii-iii) were performed. (Ei) Yes1 architecture domain and its GC-related mutants in GC patients. (Eii) Relative activity of Yes1 was analyzed. (Eiii) IP kinase assay. Briefly, Yes1 was immunoprecipitated from HEK293 cells expressing Flag-tagged WT-Yes1 or its mutants. Then the immunoprecipitated Yes1 was mixed with [ 32 P] ATP and a known substrate peptide of Yes1. The results were normalized to 1.0 for WT-Yes1. (Fi-ii) The effects of GC-related Yes1 mutants on GC cells. (Gi-ii) The invasion and migration of GC cells were affected by GC-associated Yes1 mutants. Data are shown as mean ± SEM; **P < 0.01; ***P < 0.001.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Western Blot, Infection, Activity Assay, Quantitative RT-PCR, IP-Kinase Assay, Immunoprecipitation, Expressing, Migration

    (A) Establishing WT-Cul9 and Cul9-Y1505D knockin mice. Schematic genome maps of wild-type and targeted alleles were shown. Loxp sites were shown. (B) Representative genotyping PCR result. (C) Sequencing confirmed mutant site. (D) Scatter plots showing the mass (Di) of mice stomachs and gastric tumors, and the incidence (Dii) of tumors at 20-week-old age. (E) Representative H&E-staining and tumor pictures at 20-week-old age. (F) Mice survival curve. (G) The metabolic profiling in SGC7901 cells as indicated. (H) ATAC sequence of SGC7901 cells as indicated. (I) Cul9-Y1505D and Cul9-Y1505F mutants differently affected RNA and DNA biosynthesis in SGC7901 cells, respectively. (J) The Yes1 inhibitor rescued the phenotype of Cul9 loss in SGC7901 cells. (K) Leflunomide and Mycophenolate mofetil (MMF), two inhibitors targeting purine and pyrimidine synthesis pathways, respectively, inhibited the growth of SGC7901 cells expressing Cul9-Y1505D. Data are shown as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: (A) Establishing WT-Cul9 and Cul9-Y1505D knockin mice. Schematic genome maps of wild-type and targeted alleles were shown. Loxp sites were shown. (B) Representative genotyping PCR result. (C) Sequencing confirmed mutant site. (D) Scatter plots showing the mass (Di) of mice stomachs and gastric tumors, and the incidence (Dii) of tumors at 20-week-old age. (E) Representative H&E-staining and tumor pictures at 20-week-old age. (F) Mice survival curve. (G) The metabolic profiling in SGC7901 cells as indicated. (H) ATAC sequence of SGC7901 cells as indicated. (I) Cul9-Y1505D and Cul9-Y1505F mutants differently affected RNA and DNA biosynthesis in SGC7901 cells, respectively. (J) The Yes1 inhibitor rescued the phenotype of Cul9 loss in SGC7901 cells. (K) Leflunomide and Mycophenolate mofetil (MMF), two inhibitors targeting purine and pyrimidine synthesis pathways, respectively, inhibited the growth of SGC7901 cells expressing Cul9-Y1505D. Data are shown as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Knock-In, Sequencing, Mutagenesis, Staining, Expressing

    (A) IHC data. (B) The Pearson correlation assays as indicated (n =101). (C) Kaplan-Meier assays based on Cul9 and Yes1 levels in patients with GC (n=110). (D) Schematic model about the role of Yes1-Cul9 feedback axis in GC development.

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet: (A) IHC data. (B) The Pearson correlation assays as indicated (n =101). (C) Kaplan-Meier assays based on Cul9 and Yes1 levels in patients with GC (n=110). (D) Schematic model about the role of Yes1-Cul9 feedback axis in GC development.

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques:

    Journal: bioRxiv

    Article Title: Yes1-mediated Cul9 phosphorylation promotes the metabolic reprogramming in gastric cancer

    doi: 10.1101/2023.10.18.562906

    Figure Lengend Snippet:

    Article Snippet: Their sequence was as follows: siCul9- #1: 5′-GCUGAGAGACACGUUGUUUAG-3′; siCul9- #2: 5′-UACUGAGGGUGCUCUUCUG-3′; or scramble : 5′-GAUGCUCGCACAGCACAAU-3′; shYes1-#1 : 5′-CCAGCCTACATTCACTTCTAA-3′ and shYes1-#2: 5′-CCTCGAGAATCTTTGCGACTA-3′; shcontrol : 5′-CCGCAGGTATGCACGCGT-3′. shp62 : 5’-TAGTACAACTGCTAGTTATTT-3’; the targeting sequence of BECN1 is 5’-CCGACTTGTTCCTTACGGAAA-3’, and that of OPTN is< 5′-GCACGGCATCGTCTAAATA-3′. siRNA against MBD2: 5’-GAAGAUGAUGCCAGUAAUUUU-3′ and its control: 5′-UUCUCCGAACGUGUCACGUTT-3′. siCOP1 and shRNAs against Cul9 and ATG5 were from Origene, shCOP1 was from Santa Cruz Biotechnology, and siRNA against Yes1 was from Invitrogen.

    Techniques: Expressing

    Figure 1. Interaction with YES1 decreases the PM abundance and function of rF508del-CFTR in airway cells. (A) Intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR (r∆F, after rescue to the PM by 48 h treatment with 5 µM VX-661), were labelled with either an anti-Flag antibody or a non-specific IgG prior to non-denaturing lysis, and antibody-bound CFTR complexes were precipitated using protein G-coupled magnetic beads. Input protein levels were adjusted so that equivalent amounts of wt- and rF508del-CFTR were precipitated. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YES1. Tubulin (known to not interact with CFTR at the PM [24]) was used as an additional co-precipitation control. (B) Efficiency of siRNA-mediated depletion in CFBE cells expressing mCherry-F508del-CFTR, rescued as in (A), transfected with either a mock siRNA (siCtrl) or a commercial triple oligonucleotide mix against YES1 (siYES1). Representative WBs of CFTR, YES1, and tubulin (used as loading control) are shown (left panels), as well as the quantifications as means ± SEM from four independent experiments (right panel). (C) Immunofluorescence images of CFBE cells expressing Flag-tagged mCherry-F508del-CFTR rescued to the PM by 48 h treatment with 5 µM VX-661. The cells were transfected as in (B) and treated for 3 h with either vehicle (DMSO) or 10 µM of YES1 inhibitor SU6656. rF508del-CFTR at the surface of intact cells was immunolabelled on ice using an anti-Flag antibody followed by an Alexa 488-conjugated secondary antibody. Cells were then fixed, and the nuclei were stained with DAPI. Confocal images of the cells’ surface, showing surface CFTR staining in green (left panels) and a merged overlay image of surface (green) and total CFTR signals (mCherry, red) along with the stained nuclei (DAPI, blue) are shown in the right panels. White scale bars represent 10 µm. (D) Representative traces of ion transport activity measured through iodide-induced HS-YFP sensor fluorescence decay of untagged F508del-CFTR CFBE cells treated with 5 µM VX-661 for 48 h. The cells were transfected and treated as in (C) and co-treated with or without 25 µM of inh172 15 min prior to stimulation for 30 min in PBS with Fsk (5 µM) and Gen (10 µM), in the presence or absence of inh172. This was followed by continuous fluorescence recording and the addition of I- (represented by the black arrow, final concentration 100 mM). (E) Quantification of HS-YFP fluorescence quenching rates (QR) of at least five independent assays for each condition, calculated by fitting the iodide assay results to exponential decay curves. The means ± SEM are shown. ns—not significant, ** p < 0.01, and *** p < 0.001 between conditions indicated by the horizontal lines.

    Journal: Biomolecules

    Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

    doi: 10.3390/biom13060949

    Figure Lengend Snippet: Figure 1. Interaction with YES1 decreases the PM abundance and function of rF508del-CFTR in airway cells. (A) Intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR (r∆F, after rescue to the PM by 48 h treatment with 5 µM VX-661), were labelled with either an anti-Flag antibody or a non-specific IgG prior to non-denaturing lysis, and antibody-bound CFTR complexes were precipitated using protein G-coupled magnetic beads. Input protein levels were adjusted so that equivalent amounts of wt- and rF508del-CFTR were precipitated. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YES1. Tubulin (known to not interact with CFTR at the PM [24]) was used as an additional co-precipitation control. (B) Efficiency of siRNA-mediated depletion in CFBE cells expressing mCherry-F508del-CFTR, rescued as in (A), transfected with either a mock siRNA (siCtrl) or a commercial triple oligonucleotide mix against YES1 (siYES1). Representative WBs of CFTR, YES1, and tubulin (used as loading control) are shown (left panels), as well as the quantifications as means ± SEM from four independent experiments (right panel). (C) Immunofluorescence images of CFBE cells expressing Flag-tagged mCherry-F508del-CFTR rescued to the PM by 48 h treatment with 5 µM VX-661. The cells were transfected as in (B) and treated for 3 h with either vehicle (DMSO) or 10 µM of YES1 inhibitor SU6656. rF508del-CFTR at the surface of intact cells was immunolabelled on ice using an anti-Flag antibody followed by an Alexa 488-conjugated secondary antibody. Cells were then fixed, and the nuclei were stained with DAPI. Confocal images of the cells’ surface, showing surface CFTR staining in green (left panels) and a merged overlay image of surface (green) and total CFTR signals (mCherry, red) along with the stained nuclei (DAPI, blue) are shown in the right panels. White scale bars represent 10 µm. (D) Representative traces of ion transport activity measured through iodide-induced HS-YFP sensor fluorescence decay of untagged F508del-CFTR CFBE cells treated with 5 µM VX-661 for 48 h. The cells were transfected and treated as in (C) and co-treated with or without 25 µM of inh172 15 min prior to stimulation for 30 min in PBS with Fsk (5 µM) and Gen (10 µM), in the presence or absence of inh172. This was followed by continuous fluorescence recording and the addition of I- (represented by the black arrow, final concentration 100 mM). (E) Quantification of HS-YFP fluorescence quenching rates (QR) of at least five independent assays for each condition, calculated by fitting the iodide assay results to exponential decay curves. The means ± SEM are shown. ns—not significant, ** p < 0.01, and *** p < 0.001 between conditions indicated by the horizontal lines.

    Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Expressing, Lysis, Magnetic Beads, Control, Transfection, Staining, Activity Assay, Concentration Assay

    Figure 2. YES1 inhibition increases rF508del-CFTR retention at the PM upon thermal destabilization. (A) Diagram depicting the parallel cell surface protein biotinylation assays used to assess rF508del- CFTR thermal stability and internalization. Replicate dishes of CFBE cells expressing F508del-CFTR were incubated for 48 h at 30 ◦C in the presence of 5 µM of VX-661. One of the replicates was directly placed on ice, then the surface proteins were labeled with sulfo-NHS-SS-biotin, lysed, and the surface-labeled proteins were captured using streptavidin beads. These precipitates represented the input amount of CFTR at the PM without any thermal destabilization (DMSO 30 ◦C). A second set of replicates were moved to 37 ◦C in the absence (DMSO) or presence of YES1 inhibitors (10 µM SU6656 or 1 µM P505-15). Three hours later, surface proteins were labelled and isolated, as described above. This second set revealed the amount of CFTR remaining at the PM after thermal destabilization (TS) and inhibitor treatment. A third set of replicates was first labeled with biotin, then placed at 37 ◦C for 3 h in the presence or absence of YES inhibitors. These samples were then returned to ice, and the labeled CFTR remaining at the cell surface was stripped from biotin with glutathione prior to lysis and isolation of the labeled proteins that entered the cells. This third set represents the amount of CFTR internalized from the surface upon TS and inhibitor treatment. (B) Analysis of input lysates and biotin-labelled fractions obtained as described in (A). WBs representative of five independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut- 1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. (C) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in (B) after normalization to Glut-1 levels and to the respective controls. *** p < 0.001 relative to DMSO (30 ◦C), ## p < 0.01 and ### p < 0.001, both relative to DMSO in the internalized set.

    Journal: Biomolecules

    Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

    doi: 10.3390/biom13060949

    Figure Lengend Snippet: Figure 2. YES1 inhibition increases rF508del-CFTR retention at the PM upon thermal destabilization. (A) Diagram depicting the parallel cell surface protein biotinylation assays used to assess rF508del- CFTR thermal stability and internalization. Replicate dishes of CFBE cells expressing F508del-CFTR were incubated for 48 h at 30 ◦C in the presence of 5 µM of VX-661. One of the replicates was directly placed on ice, then the surface proteins were labeled with sulfo-NHS-SS-biotin, lysed, and the surface-labeled proteins were captured using streptavidin beads. These precipitates represented the input amount of CFTR at the PM without any thermal destabilization (DMSO 30 ◦C). A second set of replicates were moved to 37 ◦C in the absence (DMSO) or presence of YES1 inhibitors (10 µM SU6656 or 1 µM P505-15). Three hours later, surface proteins were labelled and isolated, as described above. This second set revealed the amount of CFTR remaining at the PM after thermal destabilization (TS) and inhibitor treatment. A third set of replicates was first labeled with biotin, then placed at 37 ◦C for 3 h in the presence or absence of YES inhibitors. These samples were then returned to ice, and the labeled CFTR remaining at the cell surface was stripped from biotin with glutathione prior to lysis and isolation of the labeled proteins that entered the cells. This third set represents the amount of CFTR internalized from the surface upon TS and inhibitor treatment. (B) Analysis of input lysates and biotin-labelled fractions obtained as described in (A). WBs representative of five independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut- 1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. (C) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in (B) after normalization to Glut-1 levels and to the respective controls. *** p < 0.001 relative to DMSO (30 ◦C), ## p < 0.01 and ### p < 0.001, both relative to DMSO in the internalized set.

    Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Inhibition, Expressing, Incubation, Labeling, Isolation, Lysis

    Figure 3. YAP1 is required for the binding of YES1 to rF508del-CFTR complexes at the PM. (A) STRING- based analysis (https://string-db.org/, accessed on 19 October 2021) of the strength of annotated evidence on the interaction between YES1 and the CFTR/NHERF1 (SLC9A3R1)/Ezrin (EZR) mem- brane anchoring complex (the thickness of the grey lines is proportional to the degree of confidence for the interaction between the two proteins they connect, extrapolated from text mining, experi- mental, and database-collected evidence). (B) A STRING-generated expanded interaction network, extended (green nodes) around the core complex in (A) (red nodes). (C) CFTR-containing complexes were immunoprecipitated from the PM of intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR, as described for Figure 1A. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YAP1. Tubulin was used as an additional co-precipitation control. (D) CFBE cells expressing untagged F508del-CFTR transfected with either mock siRNA (siCtrl) or a commercial siRNA mix targeting YAP1 (siYAP1), were incubated with 5 µM of VX-661 for 48 h at 30 ◦C to coax most of the mutant channel to the PM. The cells were then lysed in non-denaturing conditions and YES1 immunoprecipitated with a specific antibody (a non-specific IgG was used as a control) from whole cell lysates (WCL). Both input lysates and co-precipitates were analyzed using WB to assess the levels of precipitated YES1 and co-precipitated rF508del-CFTR. (E) Thermal shift (TS) assay, as described in Figure 2, to assess the thermal stability of untagged rF508del-CFTR in CFBE cells transfected with mock siRNA (siCtrl) or one of two commercial siRNA mixes targeting either YAP1 (siYAP1) or YES1 (siYES1). WBs repre- sentative of at least four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. (F) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in (E) after normalization to Glut-1 levels and to siCtrl (30 ◦C). *** p < 0.001 relative to siCtrl (30 ◦C), ## p < 0.01 and ### p < 0.001, both relative to siCtrl (TS).

    Journal: Biomolecules

    Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

    doi: 10.3390/biom13060949

    Figure Lengend Snippet: Figure 3. YAP1 is required for the binding of YES1 to rF508del-CFTR complexes at the PM. (A) STRING- based analysis (https://string-db.org/, accessed on 19 October 2021) of the strength of annotated evidence on the interaction between YES1 and the CFTR/NHERF1 (SLC9A3R1)/Ezrin (EZR) mem- brane anchoring complex (the thickness of the grey lines is proportional to the degree of confidence for the interaction between the two proteins they connect, extrapolated from text mining, experi- mental, and database-collected evidence). (B) A STRING-generated expanded interaction network, extended (green nodes) around the core complex in (A) (red nodes). (C) CFTR-containing complexes were immunoprecipitated from the PM of intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR, as described for Figure 1A. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YAP1. Tubulin was used as an additional co-precipitation control. (D) CFBE cells expressing untagged F508del-CFTR transfected with either mock siRNA (siCtrl) or a commercial siRNA mix targeting YAP1 (siYAP1), were incubated with 5 µM of VX-661 for 48 h at 30 ◦C to coax most of the mutant channel to the PM. The cells were then lysed in non-denaturing conditions and YES1 immunoprecipitated with a specific antibody (a non-specific IgG was used as a control) from whole cell lysates (WCL). Both input lysates and co-precipitates were analyzed using WB to assess the levels of precipitated YES1 and co-precipitated rF508del-CFTR. (E) Thermal shift (TS) assay, as described in Figure 2, to assess the thermal stability of untagged rF508del-CFTR in CFBE cells transfected with mock siRNA (siCtrl) or one of two commercial siRNA mixes targeting either YAP1 (siYAP1) or YES1 (siYES1). WBs repre- sentative of at least four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. (F) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in (E) after normalization to Glut-1 levels and to siCtrl (30 ◦C). *** p < 0.001 relative to siCtrl (30 ◦C), ## p < 0.01 and ### p < 0.001, both relative to siCtrl (TS).

    Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Binding Assay, Generated, Immunoprecipitation, Expressing, Control, Transfection, Incubation, Mutagenesis

    Figure 4. The MAPK pathway participates in YES1-mediated internalization of rF508del-CFTR at the PM. The thermal shift (TS) assay, as described in Figure 2, was used to assess the thermal stability of untagged rF508del-CFTR in CFBE cells. (A) The cells were treated for 3 h with either vehicle (DMSO) or 10 µM of selumetinib, or (C) transfected with empty vector or Myc-H-RAS-V12 (HRAS) and then treated for 3 h with vehicle (DMSO) or 10 µM of SU6656, as indicated. WBs representative of input and cell surface fractions from four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. H-RAS V12 was detected using an anti-Myc antibody, and an anti-phosphorylated ERK1/2 antibody was used to monitor MAPK path- way activity, which was quantified and shown below the respective blot lanes. (B,D) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from four independent assays after normalization to Glut-1 levels and DMSO (30 ◦C). * p < 0.05, ** p < 0.01, and *** p < 0.001, relative to DMSO (30 ◦C) in (B), and as indicated by the horizontal lines in (C); ## p < 0.01, relative to DMSO (TS) in (B).

    Journal: Biomolecules

    Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

    doi: 10.3390/biom13060949

    Figure Lengend Snippet: Figure 4. The MAPK pathway participates in YES1-mediated internalization of rF508del-CFTR at the PM. The thermal shift (TS) assay, as described in Figure 2, was used to assess the thermal stability of untagged rF508del-CFTR in CFBE cells. (A) The cells were treated for 3 h with either vehicle (DMSO) or 10 µM of selumetinib, or (C) transfected with empty vector or Myc-H-RAS-V12 (HRAS) and then treated for 3 h with vehicle (DMSO) or 10 µM of SU6656, as indicated. WBs representative of input and cell surface fractions from four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. H-RAS V12 was detected using an anti-Myc antibody, and an anti-phosphorylated ERK1/2 antibody was used to monitor MAPK path- way activity, which was quantified and shown below the respective blot lanes. (B,D) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from four independent assays after normalization to Glut-1 levels and DMSO (30 ◦C). * p < 0.05, ** p < 0.01, and *** p < 0.001, relative to DMSO (30 ◦C) in (B), and as indicated by the horizontal lines in (C); ## p < 0.01, relative to DMSO (TS) in (B).

    Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Transfection, Plasmid Preparation, Activity Assay

    Figure 5. SHC1 phosphorylation by YES1 mediates rF508del-CFTR internalization via MAPK path- way signaling. (A) Effects of YES1 inhibitors. Lysates from F508del-CFTR expressing CFBE cells were incubated with 5 µM of VX-661 for 48 h at 30 ◦C, treated with either vehicle (DMSO), SU6656 (10 µM), or P505-15 (1 µM) for 3 h at 37 ◦C, then were analyzed using WB. Immunoblots representa- tive of three independent experiments, probed with antibodies against the indicated proteins, are shown. p-SHC1 indicates the level of SHC1 phosphorylation at Tyr239/240 in the different conditions, and an anti-phosphorylated ERK1/2 antibody (p-ERK1/2) was used to monitor MAPK pathway activity (both show quantified band intensities below their respective blots). (B) Thermal shift (TS) assay described in Figure 2 to assess how much rF508del-CFTR remained at the PM after thermal destabilization in CFBE cells transfected either with a mock siRNA (siCtrl) or a siRNA against SHC1 (siSHC1). WBs representative of input and cell surface fractions, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. Quantifications of SCH1 depletion efficiency and ERK1/2 phosphorylation levels are shown below their respective blots. (C) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from three independent assays after normalization to Glut-1 levels and to siCtrl (30 ◦C). *** p < 0.001 relative to siCtrl (30 ◦C), ## p < 0.01 relative to siCtrl (TS).

    Journal: Biomolecules

    Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

    doi: 10.3390/biom13060949

    Figure Lengend Snippet: Figure 5. SHC1 phosphorylation by YES1 mediates rF508del-CFTR internalization via MAPK path- way signaling. (A) Effects of YES1 inhibitors. Lysates from F508del-CFTR expressing CFBE cells were incubated with 5 µM of VX-661 for 48 h at 30 ◦C, treated with either vehicle (DMSO), SU6656 (10 µM), or P505-15 (1 µM) for 3 h at 37 ◦C, then were analyzed using WB. Immunoblots representa- tive of three independent experiments, probed with antibodies against the indicated proteins, are shown. p-SHC1 indicates the level of SHC1 phosphorylation at Tyr239/240 in the different conditions, and an anti-phosphorylated ERK1/2 antibody (p-ERK1/2) was used to monitor MAPK pathway activity (both show quantified band intensities below their respective blots). (B) Thermal shift (TS) assay described in Figure 2 to assess how much rF508del-CFTR remained at the PM after thermal destabilization in CFBE cells transfected either with a mock siRNA (siCtrl) or a siRNA against SHC1 (siSHC1). WBs representative of input and cell surface fractions, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. Quantifications of SCH1 depletion efficiency and ERK1/2 phosphorylation levels are shown below their respective blots. (C) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from three independent assays after normalization to Glut-1 levels and to siCtrl (30 ◦C). *** p < 0.001 relative to siCtrl (30 ◦C), ## p < 0.01 relative to siCtrl (TS).

    Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Phospho-proteomics, Expressing, Incubation, Western Blot, Activity Assay, Transfection

    Figure 6. Proposed model for SHC1-mediated removal of CFTR from the PM through activation of the MAPK pathway. (A) Phosphorylation of PM-anchored wt-CFTR at Tyr512 by SYK kinase leads to its recognition and binding by the adaptor protein SHC1. This links CFTR internalization to the activation of the MAPK pathway downstream of receptor tyrosine kinases. (B) F508del-CFTR pharmacologically rescued to the PM is not phosphorylated by SYK, but its deficient anchoring to the actin cytoskeleton allows its interaction with the YES1 kinase via the adaptor protein YAP1 and the scaffold protein NHERF1. SHC1 is a substrate for YES1 at the PM, and its phosphorylation by YES1 increases its affinity to membrane receptors in the vicinity, enhancing their activation of the MAPK pathway. This could contribute to the much faster internalization rate of rF508del-CFTR compared to the wild-type protein. RTK—receptor tyrosine kinases; EB—Ezrin binding domain; 1-2—NHERF1’s PDZ1 and PDZ2.

    Journal: Biomolecules

    Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

    doi: 10.3390/biom13060949

    Figure Lengend Snippet: Figure 6. Proposed model for SHC1-mediated removal of CFTR from the PM through activation of the MAPK pathway. (A) Phosphorylation of PM-anchored wt-CFTR at Tyr512 by SYK kinase leads to its recognition and binding by the adaptor protein SHC1. This links CFTR internalization to the activation of the MAPK pathway downstream of receptor tyrosine kinases. (B) F508del-CFTR pharmacologically rescued to the PM is not phosphorylated by SYK, but its deficient anchoring to the actin cytoskeleton allows its interaction with the YES1 kinase via the adaptor protein YAP1 and the scaffold protein NHERF1. SHC1 is a substrate for YES1 at the PM, and its phosphorylation by YES1 increases its affinity to membrane receptors in the vicinity, enhancing their activation of the MAPK pathway. This could contribute to the much faster internalization rate of rF508del-CFTR compared to the wild-type protein. RTK—receptor tyrosine kinases; EB—Ezrin binding domain; 1-2—NHERF1’s PDZ1 and PDZ2.

    Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Membrane

    Effects of cGAS/STING knockdown on the migration and invasion of RA FLSs induced by cytosolic dsDNA. RA FLSs were transfected with siRNA or ISD dsDNA (2 µg/mL). (A) RA FLSs were transfected with cGAS or STING siRNA (sicGAS1-3 or siSTING1-3) or control siRNA (siC) for 48 hours and subjected to qRT-PCR analysis for mRNA expression or western blot analysis for protein expression. A representative blot of at least 3 independent experiments is shown. (B,C) Migration and invasion of RA FLSs were measured using a Transwell assay. Invasion was evaluated using inserts coated with matrigel basement membrane matrix. The relative migratory or invasive rate was calculated by counting migrated or invaded cells and then normalized to the siC or siC + dsDNA. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. The graphs show the relative migration rates. (D) cGAS or STING knockdown impaired the lamellipodia formation in RA FLSs. RA FLSs were wounded and stimulated with dsDNA (2 µg/mL) for 8 hours. Representative images are shown (original magnification: 400×). The yellow arrow indicates lamellipodia formation. The data (A-C) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. siC; #P<0.05; ##P<0.01 vs. siC + dsDNA. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; sicGAS, cGAS siRNA; siSTING, STING siRNA; siC, control siRNA; dsDNA, double-stranded DNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Journal: Annals of Translational Medicine

    Article Title: cGAS/STING signaling in the regulation of rheumatoid synovial aggression

    doi: 10.21037/atm-21-4533

    Figure Lengend Snippet: Effects of cGAS/STING knockdown on the migration and invasion of RA FLSs induced by cytosolic dsDNA. RA FLSs were transfected with siRNA or ISD dsDNA (2 µg/mL). (A) RA FLSs were transfected with cGAS or STING siRNA (sicGAS1-3 or siSTING1-3) or control siRNA (siC) for 48 hours and subjected to qRT-PCR analysis for mRNA expression or western blot analysis for protein expression. A representative blot of at least 3 independent experiments is shown. (B,C) Migration and invasion of RA FLSs were measured using a Transwell assay. Invasion was evaluated using inserts coated with matrigel basement membrane matrix. The relative migratory or invasive rate was calculated by counting migrated or invaded cells and then normalized to the siC or siC + dsDNA. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. The graphs show the relative migration rates. (D) cGAS or STING knockdown impaired the lamellipodia formation in RA FLSs. RA FLSs were wounded and stimulated with dsDNA (2 µg/mL) for 8 hours. Representative images are shown (original magnification: 400×). The yellow arrow indicates lamellipodia formation. The data (A-C) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. siC; #P<0.05; ##P<0.01 vs. siC + dsDNA. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; sicGAS, cGAS siRNA; siSTING, STING siRNA; siC, control siRNA; dsDNA, double-stranded DNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Article Snippet: cGAS small interfering RNA (siRNA), STING siRNA, MST1 siRNA, FOXO1 siRNA, and yes-associated protein ( YAP ) siRNA were purchased from Ribobio (Guangzhou, China).

    Techniques: Knockdown, Migration, Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, Transwell Assay, Membrane, Staining, Reverse Transcription, Polymerase Chain Reaction

    The effect of cGAS or STING inhibition on the invasion of RA FLSs into human cartilage implants transferred under the skin of SCID mice. (A) RA FLSs were transfected with cGAS or STING shRNA (shcGAS1-3 or shSTING1-3) or control vectors for 48 hours and subjected to qRT-PCR analysis for mRNA expression or western blot analysis for protein expression. A representative blot of 3 independent experiments is shown. (B) RA FLSs, transfected with vector, cGAS or STING shRNA were transferred side by side into the left or right flanks of the SCID mouse model (n=6 for each group). The mice were killed after 50 days, and implants were stained with haematoxylin and histologically evaluated for cell invasion. The arrows indicate RA FLSs invading Ca (upper image; original magnification: 200×). Data represent the mean mean ± SEM of semiquantitative analysis. *P<0.05; **P<0.01 vs. vector. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; sicGAS, cGAS siRNA; siSTING, STING siRNA; siC, control siRNA; dsDNA, double-stranded DNA; Ca, cartilage; SCID, severe combined immunodeficiency; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Journal: Annals of Translational Medicine

    Article Title: cGAS/STING signaling in the regulation of rheumatoid synovial aggression

    doi: 10.21037/atm-21-4533

    Figure Lengend Snippet: The effect of cGAS or STING inhibition on the invasion of RA FLSs into human cartilage implants transferred under the skin of SCID mice. (A) RA FLSs were transfected with cGAS or STING shRNA (shcGAS1-3 or shSTING1-3) or control vectors for 48 hours and subjected to qRT-PCR analysis for mRNA expression or western blot analysis for protein expression. A representative blot of 3 independent experiments is shown. (B) RA FLSs, transfected with vector, cGAS or STING shRNA were transferred side by side into the left or right flanks of the SCID mouse model (n=6 for each group). The mice were killed after 50 days, and implants were stained with haematoxylin and histologically evaluated for cell invasion. The arrows indicate RA FLSs invading Ca (upper image; original magnification: 200×). Data represent the mean mean ± SEM of semiquantitative analysis. *P<0.05; **P<0.01 vs. vector. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; sicGAS, cGAS siRNA; siSTING, STING siRNA; siC, control siRNA; dsDNA, double-stranded DNA; Ca, cartilage; SCID, severe combined immunodeficiency; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Article Snippet: cGAS small interfering RNA (siRNA), STING siRNA, MST1 siRNA, FOXO1 siRNA, and yes-associated protein ( YAP ) siRNA were purchased from Ribobio (Guangzhou, China).

    Techniques: Inhibition, Transfection, shRNA, Control, Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, Staining, Reverse Transcription, Polymerase Chain Reaction

    The effect of cGAS/STING inhibition on MST1 activation. (A) The effect of ISD dsDNA transfection on MST1 phosphorylation. RA FLSs were transfected with ISD dsDNA (2 µg/mL) for different times (2, 4, 6, and 8 hours). Western blotting was used to detect the expression of total and phosphorylated MST1. (B) RA FLSs, transfected with cGAS or STING siRNA, or control siRNA, were serum-starved overnight and then transfected with dsDNA (2 µg/mL) for 2 hours. Representative images of immunoblots (left panel) and densitometric quantification (right panel) of phosphorylated MST1 expression are shown. (C) Efficiency of MST1 knockdown. RA FLSs were transfected with MST1 siRNA (siMST1-1-3) or siC for 48 hours and subjected to qRT-PCR analysis and western blot analysis. (D) The effect of MST1 knockdown on migration and invasion. RA FLSs, transfected with MST1 siRNA, were serum-starved overnight and then transfected with ISD dsDNA (2 µg/mL) for 8 hours. Migration and invasion were measured using a Boyden chamber. The relative migration and invasion rate was calculated by counting migrated or invaded cells and was followed by normalization to the siC. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. Graphs show the relative migratory and invasive rates. (E) The effect of MST1 knockdown on lamellipodia formation of RA FLSs. Representative images are shown (original magnification: 400×). The yellow arrows indicate lamellipodia formation. A representative blot of at least 3 independent experiments is shown. The data (A-D) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. siC; #P<0.05; ##P<0.01 vs. siC + dsDNA. MST1, mammalian sterile 20-like kinase 1; RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; sicGAS, cGAS siRNA; siSTING, STING siRNA; siC, control siRNA; dsDNA, double-stranded DNA; siMST1, MST1 siRNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Journal: Annals of Translational Medicine

    Article Title: cGAS/STING signaling in the regulation of rheumatoid synovial aggression

    doi: 10.21037/atm-21-4533

    Figure Lengend Snippet: The effect of cGAS/STING inhibition on MST1 activation. (A) The effect of ISD dsDNA transfection on MST1 phosphorylation. RA FLSs were transfected with ISD dsDNA (2 µg/mL) for different times (2, 4, 6, and 8 hours). Western blotting was used to detect the expression of total and phosphorylated MST1. (B) RA FLSs, transfected with cGAS or STING siRNA, or control siRNA, were serum-starved overnight and then transfected with dsDNA (2 µg/mL) for 2 hours. Representative images of immunoblots (left panel) and densitometric quantification (right panel) of phosphorylated MST1 expression are shown. (C) Efficiency of MST1 knockdown. RA FLSs were transfected with MST1 siRNA (siMST1-1-3) or siC for 48 hours and subjected to qRT-PCR analysis and western blot analysis. (D) The effect of MST1 knockdown on migration and invasion. RA FLSs, transfected with MST1 siRNA, were serum-starved overnight and then transfected with ISD dsDNA (2 µg/mL) for 8 hours. Migration and invasion were measured using a Boyden chamber. The relative migration and invasion rate was calculated by counting migrated or invaded cells and was followed by normalization to the siC. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. Graphs show the relative migratory and invasive rates. (E) The effect of MST1 knockdown on lamellipodia formation of RA FLSs. Representative images are shown (original magnification: 400×). The yellow arrows indicate lamellipodia formation. A representative blot of at least 3 independent experiments is shown. The data (A-D) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. siC; #P<0.05; ##P<0.01 vs. siC + dsDNA. MST1, mammalian sterile 20-like kinase 1; RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; sicGAS, cGAS siRNA; siSTING, STING siRNA; siC, control siRNA; dsDNA, double-stranded DNA; siMST1, MST1 siRNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Article Snippet: cGAS small interfering RNA (siRNA), STING siRNA, MST1 siRNA, FOXO1 siRNA, and yes-associated protein ( YAP ) siRNA were purchased from Ribobio (Guangzhou, China).

    Techniques: Inhibition, Activation Assay, Transfection, Phospho-proteomics, Western Blot, Expressing, Control, Knockdown, Quantitative RT-PCR, Migration, Membrane, Staining, Sterility, Reverse Transcription, Polymerase Chain Reaction

    The effects of MST1 knockdown on canonical LATS1 and YAP protein expression in RA FLSs. RA FLSs, transfected with cGAS or STING siRNA or control siRNA, were serum-starved overnight and transfected with dsDNA (2 µg/mL) for 8 hours. (A) LATS1 and (B) YAP protein expression was measured using western blotting. Densitometric quantification of protein levels (right panel) is shown as the mean ± SEM from at least 3 independent experiments. LATS1, large tumor suppressor kinase 1; YAP, yes-associated protein; RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; MST1, mammalian sterile 20-like kinase 1; siMST1, MST1 siRNA; dsDNA, double-stranded DNA.

    Journal: Annals of Translational Medicine

    Article Title: cGAS/STING signaling in the regulation of rheumatoid synovial aggression

    doi: 10.21037/atm-21-4533

    Figure Lengend Snippet: The effects of MST1 knockdown on canonical LATS1 and YAP protein expression in RA FLSs. RA FLSs, transfected with cGAS or STING siRNA or control siRNA, were serum-starved overnight and transfected with dsDNA (2 µg/mL) for 8 hours. (A) LATS1 and (B) YAP protein expression was measured using western blotting. Densitometric quantification of protein levels (right panel) is shown as the mean ± SEM from at least 3 independent experiments. LATS1, large tumor suppressor kinase 1; YAP, yes-associated protein; RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; MST1, mammalian sterile 20-like kinase 1; siMST1, MST1 siRNA; dsDNA, double-stranded DNA.

    Article Snippet: cGAS small interfering RNA (siRNA), STING siRNA, MST1 siRNA, FOXO1 siRNA, and yes-associated protein ( YAP ) siRNA were purchased from Ribobio (Guangzhou, China).

    Techniques: Knockdown, Expressing, Transfection, Control, Western Blot, Sterility

    The effect of MST1 knockdown on FOXO1 activation. (A) The effect of ISD dsDNA treatment on FOXO1 phosphorylation. RA FLSs were transfected with ISD dsDNA (2 mg/mL) for different times (2, 4, 6, and 8 hours). Protein expression was detected by western blot. (B) The effect of MST1 knockdown on FOXO1 phosphorylation. RA FLSs transfected with MST1 siRNA or control siRNA were serum-starved overnight and transfected with dsDNA (2 mg/mL) for 8 hours. The pFOXO1 and tFOXO1 protein expression was measured using western blotting. (C) The effect of MST1 knockdown on the nuclear translocation of FOXO1. The translocation of FOXO1 into the nucleus was evaluated using immunofluorescence staining. Representative immunofluorescent images of FOXO1 (green) and nuclei (blue) in FLSs are shown. Original magnification 400×. (D) Efficiency of FOXO1 knockdown. RA FLSs were transfected with FOXO1 siRNA (siFOXO1-1-3) or siC for 48 hours. The FOXO1 mRNA and protein expression were detected by qRT-PCR and western blotting, respectively. The effect of FOXO1 knockdown on the migration and invasion of RA FLSs. (E) RA FLSs transfected with FOXO1 siRNA were serum-starved overnight and then transfected with ISD dsDNA (2 mg/mL) for 8 hours before migration and invasion. The relative migratory and invasive rate was calculated by counting migrated or invaded cells and through normalization to the siC or siC + dsDNA. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. Graphs show the relative migration rates. The data (A,B,D,E) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. siC; #P<0.05; ##P<0.01 vs. siC + dsDNA. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; FOXO1, forkhead box 1; pFOXO1, phosphorylation of FOXO1; MST1, mammalian sterile 20-like kinase 1; siMST1, MST1 siRNA; siFOXO1, FOXO1 siRNA; siC, control siRNA; dsDNA, double-stranded DNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Journal: Annals of Translational Medicine

    Article Title: cGAS/STING signaling in the regulation of rheumatoid synovial aggression

    doi: 10.21037/atm-21-4533

    Figure Lengend Snippet: The effect of MST1 knockdown on FOXO1 activation. (A) The effect of ISD dsDNA treatment on FOXO1 phosphorylation. RA FLSs were transfected with ISD dsDNA (2 mg/mL) for different times (2, 4, 6, and 8 hours). Protein expression was detected by western blot. (B) The effect of MST1 knockdown on FOXO1 phosphorylation. RA FLSs transfected with MST1 siRNA or control siRNA were serum-starved overnight and transfected with dsDNA (2 mg/mL) for 8 hours. The pFOXO1 and tFOXO1 protein expression was measured using western blotting. (C) The effect of MST1 knockdown on the nuclear translocation of FOXO1. The translocation of FOXO1 into the nucleus was evaluated using immunofluorescence staining. Representative immunofluorescent images of FOXO1 (green) and nuclei (blue) in FLSs are shown. Original magnification 400×. (D) Efficiency of FOXO1 knockdown. RA FLSs were transfected with FOXO1 siRNA (siFOXO1-1-3) or siC for 48 hours. The FOXO1 mRNA and protein expression were detected by qRT-PCR and western blotting, respectively. The effect of FOXO1 knockdown on the migration and invasion of RA FLSs. (E) RA FLSs transfected with FOXO1 siRNA were serum-starved overnight and then transfected with ISD dsDNA (2 mg/mL) for 8 hours before migration and invasion. The relative migratory and invasive rate was calculated by counting migrated or invaded cells and through normalization to the siC or siC + dsDNA. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. Graphs show the relative migration rates. The data (A,B,D,E) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. siC; #P<0.05; ##P<0.01 vs. siC + dsDNA. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; FOXO1, forkhead box 1; pFOXO1, phosphorylation of FOXO1; MST1, mammalian sterile 20-like kinase 1; siMST1, MST1 siRNA; siFOXO1, FOXO1 siRNA; siC, control siRNA; dsDNA, double-stranded DNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Article Snippet: cGAS small interfering RNA (siRNA), STING siRNA, MST1 siRNA, FOXO1 siRNA, and yes-associated protein ( YAP ) siRNA were purchased from Ribobio (Guangzhou, China).

    Techniques: Knockdown, Activation Assay, Phospho-proteomics, Transfection, Expressing, Western Blot, Control, Translocation Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Migration, Membrane, Sterility, Reverse Transcription, Polymerase Chain Reaction

    The effect of cGAS/STING inhibition on ROS production in RA FLSs. Protein expression was measured using western blotting. (A) The effect of ISD dsDNA on ROS production. RA FLSs were transfected with ISD dsDNA (2 µg/mL) at different times (2, 4, 6, and 8 hours). Representative plots of ROS level are shown. (B) RA FLSs were pretreated with rotenone (20 nM), apocynin (30 μM), AEBSF (2 μM), carboxin (5 μM), antimycin A (2 μg/mL), and oxypurinol (0.3 mM) for 24 hours and transfected with ISD dsDNA (2 µg/mL) for 8 hours. (C) The effect of cGAS or STING knockdown on ROS production. RA FLSs transfected with cGAS, STING siRNA, or control siRNA were serum-starved overnight and then transfected with dsDNA (2 µg/mL) for 8 hours. Graphs (A-C) show the relative MFI of ROS. (D,E) The effect of rotenone or antimycin A treatment on the expression of cGAS (D), STING (D), phosphorylated MST1 (E), and phosphorylated FOXO1 (E). RA FLSs were pretreated with rotenone (20 nM) or antimycin A (2 μg/mL) for 24 hours and then transfected with ISD dsDNA (2 µg/mL) for 24 hours (cGAS and STING group), 2 hours (pMST1 group) or 8 hours (pFOXO1 group). (F) The effect of MST1 knockdown on ROS production. RA FLSs transfected with MST1 siRNA or control siRNA were serum-starved overnight and transfected with dsDNA (2 µg/mL) for 8 hours. (G) The effect of ROS production on migration and invasion. RA FLSs were pretreated with rotenone (20 nM) or antimycin A (2 μg/mL) for 24 hours and then transfected with ISD dsDNA (2 µg/mL) for 8 hours. The relative migration and invasion rate was calculated by counting migrated or invaded cells and then through normalization to the lipo or lipo + dsDNA. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. Graphs show the relative migration rates. ns indicates no statistically significant difference. The data (A-G) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. lipo; #P<0.05; ##P<0.01 vs. lipo + dsDNA. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; ROS, reactive oxygen species; LIPO, Lipofectamine 3000; CTR, control; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; MST1, mammalian sterile 20-like kinase 1; FOXO1, forkhead box 1; siC, control siRNA; sicGAS, cGAS siRNA; siSTING, STING siRNA; siMST1; MST1 siRNA; dsDNA, double-stranded DNA; ns, no statistically significant difference; MFI, mean fluorescence intensity.

    Journal: Annals of Translational Medicine

    Article Title: cGAS/STING signaling in the regulation of rheumatoid synovial aggression

    doi: 10.21037/atm-21-4533

    Figure Lengend Snippet: The effect of cGAS/STING inhibition on ROS production in RA FLSs. Protein expression was measured using western blotting. (A) The effect of ISD dsDNA on ROS production. RA FLSs were transfected with ISD dsDNA (2 µg/mL) at different times (2, 4, 6, and 8 hours). Representative plots of ROS level are shown. (B) RA FLSs were pretreated with rotenone (20 nM), apocynin (30 μM), AEBSF (2 μM), carboxin (5 μM), antimycin A (2 μg/mL), and oxypurinol (0.3 mM) for 24 hours and transfected with ISD dsDNA (2 µg/mL) for 8 hours. (C) The effect of cGAS or STING knockdown on ROS production. RA FLSs transfected with cGAS, STING siRNA, or control siRNA were serum-starved overnight and then transfected with dsDNA (2 µg/mL) for 8 hours. Graphs (A-C) show the relative MFI of ROS. (D,E) The effect of rotenone or antimycin A treatment on the expression of cGAS (D), STING (D), phosphorylated MST1 (E), and phosphorylated FOXO1 (E). RA FLSs were pretreated with rotenone (20 nM) or antimycin A (2 μg/mL) for 24 hours and then transfected with ISD dsDNA (2 µg/mL) for 24 hours (cGAS and STING group), 2 hours (pMST1 group) or 8 hours (pFOXO1 group). (F) The effect of MST1 knockdown on ROS production. RA FLSs transfected with MST1 siRNA or control siRNA were serum-starved overnight and transfected with dsDNA (2 µg/mL) for 8 hours. (G) The effect of ROS production on migration and invasion. RA FLSs were pretreated with rotenone (20 nM) or antimycin A (2 μg/mL) for 24 hours and then transfected with ISD dsDNA (2 µg/mL) for 8 hours. The relative migration and invasion rate was calculated by counting migrated or invaded cells and then through normalization to the lipo or lipo + dsDNA. Migrated and invaded cells on the membrane were stained with 0.3% crystal violet. Representative images (original magnification: 100×) are shown. Graphs show the relative migration rates. ns indicates no statistically significant difference. The data (A-G) are shown as the mean ± SEM from at least 3 independent experiments. *P<0.05; **P<0.01 vs. lipo; #P<0.05; ##P<0.01 vs. lipo + dsDNA. RA, rheumatoid arthritis; FLSs, fibroblast like-synoviocytes; ROS, reactive oxygen species; LIPO, Lipofectamine 3000; CTR, control; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; MST1, mammalian sterile 20-like kinase 1; FOXO1, forkhead box 1; siC, control siRNA; sicGAS, cGAS siRNA; siSTING, STING siRNA; siMST1; MST1 siRNA; dsDNA, double-stranded DNA; ns, no statistically significant difference; MFI, mean fluorescence intensity.

    Article Snippet: cGAS small interfering RNA (siRNA), STING siRNA, MST1 siRNA, FOXO1 siRNA, and yes-associated protein ( YAP ) siRNA were purchased from Ribobio (Guangzhou, China).

    Techniques: Inhibition, Expressing, Western Blot, Transfection, Knockdown, Control, Migration, Membrane, Staining, Sterility, Fluorescence

    A Venn diagram depicting 9 candidate common targets of miR-145-5p and linc01133. B The complementary sequence of YES1 3’UTR with miR-145-5p seed sequence predicted by TargetScan and the mutated sequence. MiR-145-5p reduced the luciferase activity of pmirGLO-YES1 3’UTR but not that with miR-145-5p binding site mutation in MKN45 ( C ) and HEK293 cells ( D ). MiR-145-5p inhibited YES1 expression in both mRNA ( E )and protein level ( F ). G YES1 3’UTR is AGO2 bounded demonstrated by RIP assay. H YES1 could be pulled down by biotin-labeled miR-145-5p. Linc01133 overexpression in HGC27 cells or knocking-down in AGS cells increased or decreased the expression of YES1 both in mRNA ( I, J ) and protein level ( K ). L Linc01133 greatly attenuated miR-145-5p induced YES1 down-regulation. Each experiment was performed in triplicates. Data are shown as mean ± SD, * P < 0.05, ** P < 0.01.

    Journal: Cell Death & Disease

    Article Title: Linc01133 contributes to gastric cancer growth by enhancing YES1-dependent YAP1 nuclear translocation via sponging miR-145-5p

    doi: 10.1038/s41419-022-04500-w

    Figure Lengend Snippet: A Venn diagram depicting 9 candidate common targets of miR-145-5p and linc01133. B The complementary sequence of YES1 3’UTR with miR-145-5p seed sequence predicted by TargetScan and the mutated sequence. MiR-145-5p reduced the luciferase activity of pmirGLO-YES1 3’UTR but not that with miR-145-5p binding site mutation in MKN45 ( C ) and HEK293 cells ( D ). MiR-145-5p inhibited YES1 expression in both mRNA ( E )and protein level ( F ). G YES1 3’UTR is AGO2 bounded demonstrated by RIP assay. H YES1 could be pulled down by biotin-labeled miR-145-5p. Linc01133 overexpression in HGC27 cells or knocking-down in AGS cells increased or decreased the expression of YES1 both in mRNA ( I, J ) and protein level ( K ). L Linc01133 greatly attenuated miR-145-5p induced YES1 down-regulation. Each experiment was performed in triplicates. Data are shown as mean ± SD, * P < 0.05, ** P < 0.01.

    Article Snippet: 2 siRNA targeting linc01133 and 3 siRNA targeting YES1 were designed by Genepharma (Shanghai, China).

    Techniques: Sequencing, Luciferase, Activity Assay, Binding Assay, Mutagenesis, Expressing, Labeling, Over Expression

    Knocking-down YES1 ameliorated linc01133 induced accelerating cell growth demonstrated by EdU assay ( A, B ) and MTS assay ( C ). D–E Colony formation assay showed decreased colony numbers formed by MKN45 cells co-transfected with pcDNA3.1-linc01133 and si-YES1. F–H The sizes, growth curves and weights of tumor xenografts showed that over-expression of linc01133 significantly enhanced tumor growth while simultaneous YES1 knocking-down inhibited tumor growth. I Ki-67 immuno-staining showed that knocking-down YES1 ameliorated linc01133 induced Ki-67 positive cells. Data are shown as mean ± SD, * P < 0.05, ** P < 0.01, N.S no significance.

    Journal: Cell Death & Disease

    Article Title: Linc01133 contributes to gastric cancer growth by enhancing YES1-dependent YAP1 nuclear translocation via sponging miR-145-5p

    doi: 10.1038/s41419-022-04500-w

    Figure Lengend Snippet: Knocking-down YES1 ameliorated linc01133 induced accelerating cell growth demonstrated by EdU assay ( A, B ) and MTS assay ( C ). D–E Colony formation assay showed decreased colony numbers formed by MKN45 cells co-transfected with pcDNA3.1-linc01133 and si-YES1. F–H The sizes, growth curves and weights of tumor xenografts showed that over-expression of linc01133 significantly enhanced tumor growth while simultaneous YES1 knocking-down inhibited tumor growth. I Ki-67 immuno-staining showed that knocking-down YES1 ameliorated linc01133 induced Ki-67 positive cells. Data are shown as mean ± SD, * P < 0.05, ** P < 0.01, N.S no significance.

    Article Snippet: 2 siRNA targeting linc01133 and 3 siRNA targeting YES1 were designed by Genepharma (Shanghai, China).

    Techniques: EdU Assay, MTS Assay, Colony Assay, Transfection, Over Expression, Immunostaining

    A , B Western and fluorescence immuno-staining showed that over-expressing linc01133 increased while knocking-down linc01133 decreased nuclear YAP1. C Simultaneous YES1 knocking-down ameliorated linc01133 induced YAP1 nuclear accumulation in MKN45 and HGC27 cells. D Western blotting showed that CDK4, CDK6 and cyclin D1 expression was in good accordance with nuclear YAP1 accumulation. E , F Immunohistochemistry staining and western blotting showed YAP1 nuclear accumulation in tumor xenografts. G Western blotting for key regulators in G1/S phase transition in tumor xenografts. Each experiment was performed in triplicates. H . Schematic illustration of the mechanisms that linc01133 promotes the growth of gastric cancer cells.

    Journal: Cell Death & Disease

    Article Title: Linc01133 contributes to gastric cancer growth by enhancing YES1-dependent YAP1 nuclear translocation via sponging miR-145-5p

    doi: 10.1038/s41419-022-04500-w

    Figure Lengend Snippet: A , B Western and fluorescence immuno-staining showed that over-expressing linc01133 increased while knocking-down linc01133 decreased nuclear YAP1. C Simultaneous YES1 knocking-down ameliorated linc01133 induced YAP1 nuclear accumulation in MKN45 and HGC27 cells. D Western blotting showed that CDK4, CDK6 and cyclin D1 expression was in good accordance with nuclear YAP1 accumulation. E , F Immunohistochemistry staining and western blotting showed YAP1 nuclear accumulation in tumor xenografts. G Western blotting for key regulators in G1/S phase transition in tumor xenografts. Each experiment was performed in triplicates. H . Schematic illustration of the mechanisms that linc01133 promotes the growth of gastric cancer cells.

    Article Snippet: 2 siRNA targeting linc01133 and 3 siRNA targeting YES1 were designed by Genepharma (Shanghai, China).

    Techniques: Western Blot, Fluorescence, Immunostaining, Expressing, Immunohistochemistry, Staining, Sublimation