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chlorothiazide  (MedChemExpress)


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    MedChemExpress chlorothiazide
    ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and <t>Chlorothiazide</t> (10 μM), or the KCC activator CLP-290 (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.
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    Images

    1) Product Images from "The WNK-OXSR1 osmosensing pathway mediates intestinal regeneration via Hippo-YAP signaling"

    Article Title: The WNK-OXSR1 osmosensing pathway mediates intestinal regeneration via Hippo-YAP signaling

    Journal: The EMBO Journal

    doi: 10.1038/s44318-026-00738-8

    ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and Chlorothiazide (10 μM), or the KCC activator CLP-290 (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.
    Figure Legend Snippet: ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and Chlorothiazide (10 μM), or the KCC activator CLP-290 (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.

    Techniques Used: Phospho-proteomics, Western Blot, Activity Assay, Triple Knockout, Double Knockout, Quantitative RT-PCR, Expressing, Two Tailed Test, Knock-Out, Generated, In Vitro, Kinase Assay, Immunoprecipitation, Purification, Isolation



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    ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and <t>Chlorothiazide</t> (10 μM), or the KCC activator CLP-290 (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.
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    ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and Chlorothiazide (10 μM), or the KCC <t>activator</t> <t>CLP-290</t> (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.
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    ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and Chlorothiazide (10 μM), or the KCC activator CLP-290 (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.

    Journal: The EMBO Journal

    Article Title: The WNK-OXSR1 osmosensing pathway mediates intestinal regeneration via Hippo-YAP signaling

    doi: 10.1038/s44318-026-00738-8

    Figure Lengend Snippet: ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and Chlorothiazide (10 μM), or the KCC activator CLP-290 (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.

    Article Snippet: The following chemicals were used in this study: Jasplakinolide (Abcam #ab141409), Latrunculin B (Abcam #ab144291), C3 transferase (Cytoskeleton #CT03-A), Staurosporine (MCE #HY-15141), Phalloidin (MCE #HY-P0028), Bumetanide (MCE #HY-17468), Chlorothiazide (MCE #HY-B0224), CLP-290 (MCE #HY-103023), WNK463 (MCE #HY-100626), and Rafoxanide (MCE #HY-17598).

    Techniques: Phospho-proteomics, Western Blot, Activity Assay, Triple Knockout, Double Knockout, Quantitative RT-PCR, Expressing, Two Tailed Test, Knock-Out, Generated, In Vitro, Kinase Assay, Immunoprecipitation, Purification, Isolation

    ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and Chlorothiazide (10 μM), or the KCC activator CLP-290 (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.

    Journal: The EMBO Journal

    Article Title: The WNK-OXSR1 osmosensing pathway mediates intestinal regeneration via Hippo-YAP signaling

    doi: 10.1038/s44318-026-00738-8

    Figure Lengend Snippet: ( A ) HEK293T cells were treated with the NKCC inhibitors Bumetanide (10 μM) and Chlorothiazide (10 μM), or the KCC activator CLP-290 (20 μM) for 2 h. Phosphorylation levels of LATS1 and YAP were analyzed by western blot. Note that OXSR1-mediated suppression of LATS1 and YAP phosphorylation is independent of NKCC, NCC, or KCC transporter activity. ( B ) Western blot was performed in the indicated HEK293T cell lines. Note that the Oxsr1; Lats1/2 triple knockout (TKO) cells, like Lats1/2 double knockout (DKO) cells, showed abolished YAP phosphorylation. ( C , D ) RT-qPCR was performed in HEK293T cells with the indicated genotypes. Note that the Oxsr1; Lats1/2 TKO cells, like Lats1/2 DKO cells, showed comparably increased expression of YAP target genes such as Ctgf ( C ) or Ankrd1 ( D ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( E ) Similar to ( B ) except for the genotypes of the cells. Note that Oxsr1; Mst1/2 TKO cells still showed higher YAP phosphorylation levels compared to Mst1/2 DKO cells. The p-YAP over total YAP levels were quantified and indicated below the blots. S.E., short exposure, L.E., long exposure. ( F , G ) Similar to ( C , D ) except for the genotypes of the cells. Note that the upregulation of YAP target genes induced by Mst1/2 double knockout was still suppressed by Oxsr1 knockout. Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( H ) Western blot was performed in the indicated HEK293A cell lines. Note that simultaneous loss of Oxsr1 , Mst1/2 , and MAP4K1/2/3/4/5/6/7 ( OMM -10KO) led to markedly reduced phosphorylation of LATS and YAP, to a level similar to that of Mst1/2 ; MAP4K1/2/3/4/5/6/7 knockout ( MM -9KO) cells. ( I , J ) RT-qPCR was performed in HEK293A cells with the indicated genotypes. Note that OMM -10KO cells, like MM -9KO cells, showed comparably increased expression of YAP target genes Ctgf ( I ) or Ankrd1 ( J ). Data were analyzed using two-tailed Student’s t test and are presented as mean ± s.d., n = 3 independent experiments. ( K ) Phosphorylation of MST1/2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note the increased phosphorylation levels of MST1/2 in Oxsr1 KO cells. ( L ) In vitro kinase assay was performed using HA-MAP4K2 immunoprecipitated from wild-type (WT) or Oxsr1 knockout (KO) HEK293A cells, and a bacterially purified GST-tagged LATS1 fragment containing its hydrophobic motif (GST-LATS1 HM ). Phosphorylation of LATS1 HM was assessed by western blot using a phospho-specific antibody against LATS1 at Threonine 1079 (p-LATS1 T1079). Note that HA-MAP4K2 isolated from Oxsr1 KO cells induced markedly higher phosphorylation of LATS1 HM compared to that from WT cells. ( M ) Phosphorylation of TAOK2 was analyzed by western blot in wild-type (WT) and Oxsr1 knockout (KO) HeLa cells. Oxsr1 KO cells generated from two independent sgRNAs were included. Note that knockout of Oxsr1 did not affect TAOK2 phosphorylation. Gel images shown are representative of at least two independent experiments.

    Article Snippet: The following chemicals were used in this study: Jasplakinolide (Abcam #ab141409), Latrunculin B (Abcam #ab144291), C3 transferase (Cytoskeleton #CT03-A), Staurosporine (MCE #HY-15141), Phalloidin (MCE #HY-P0028), Bumetanide (MCE #HY-17468), Chlorothiazide (MCE #HY-B0224), CLP-290 (MCE #HY-103023), WNK463 (MCE #HY-100626), and Rafoxanide (MCE #HY-17598).

    Techniques: Phospho-proteomics, Western Blot, Activity Assay, Triple Knockout, Double Knockout, Quantitative RT-PCR, Expressing, Two Tailed Test, Knock-Out, Generated, In Vitro, Kinase Assay, Immunoprecipitation, Purification, Isolation

    Vials of chlorothiazide sodium from lot G2590039.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Vials of chlorothiazide sodium from lot G2590039.

    Article Snippet: Intralot analysis Smoothed spectra graphs of six vials of Sagent chlorothiazide sodium from lot 230111 appear in .

    Techniques:

    Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Article Snippet: Intralot analysis Smoothed spectra graphs of six vials of Sagent chlorothiazide sodium from lot 230111 appear in .

    Techniques:

    Spectra of 204 vials of chlorothiazide sodium obtained from 28 different lots. Thirteen spectra are outliers using a full spectrum BEST from the library of 28 lots.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Spectra of 204 vials of chlorothiazide sodium obtained from 28 different lots. Thirteen spectra are outliers using a full spectrum BEST from the library of 28 lots.

    Article Snippet: Intralot analysis Smoothed spectra graphs of six vials of Sagent chlorothiazide sodium from lot 230111 appear in .

    Techniques:

    Loadings spectrum of PC 1 for the spectral library from 204 vials of chlorothiazide sodium. Important peaks are marked with arrows.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Loadings spectrum of PC 1 for the spectral library from 204 vials of chlorothiazide sodium. Important peaks are marked with arrows.

    Article Snippet: Intralot analysis Smoothed spectra graphs of six vials of Sagent chlorothiazide sodium from lot 230111 appear in .

    Techniques:

    Vials of chlorothiazide sodium from lot G2590039.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Vials of chlorothiazide sodium from lot G2590039.

    Article Snippet: Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Techniques:

    Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Article Snippet: Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Techniques:

    Spectra of 204 vials of chlorothiazide sodium obtained from 28 different lots. Thirteen spectra are outliers using a full spectrum BEST from the library of 28 lots.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Spectra of 204 vials of chlorothiazide sodium obtained from 28 different lots. Thirteen spectra are outliers using a full spectrum BEST from the library of 28 lots.

    Article Snippet: Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Techniques:

    Loadings spectrum of PC 1 for the spectral library from 204 vials of chlorothiazide sodium. Important peaks are marked with arrows.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Loadings spectrum of PC 1 for the spectral library from 204 vials of chlorothiazide sodium. Important peaks are marked with arrows.

    Article Snippet: Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Techniques:

    Vials of chlorothiazide sodium from lot G2590039.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Vials of chlorothiazide sodium from lot G2590039.

    Article Snippet: For instance, Mylan, one of the manufacturers, discontinued its chlorothiazide injection in May 2020.

    Techniques:

    Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Spectra obtained from 6 vials of Sagent chlorothiazide sodium from lot 230111.The yellow line represents vial 3 in the set and is 4.2 SDs from the remaining vials in the set of 6.

    Article Snippet: For instance, Mylan, one of the manufacturers, discontinued its chlorothiazide injection in May 2020.

    Techniques:

    Spectra of 204 vials of chlorothiazide sodium obtained from 28 different lots. Thirteen spectra are outliers using a full spectrum BEST from the library of 28 lots.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Spectra of 204 vials of chlorothiazide sodium obtained from 28 different lots. Thirteen spectra are outliers using a full spectrum BEST from the library of 28 lots.

    Article Snippet: For instance, Mylan, one of the manufacturers, discontinued its chlorothiazide injection in May 2020.

    Techniques:

    Loadings spectrum of PC 1 for the spectral library from 204 vials of chlorothiazide sodium. Important peaks are marked with arrows.

    Journal: Contact in context

    Article Title: Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials

    doi: 10.6084/m9.figshare.25773429

    Figure Lengend Snippet: Loadings spectrum of PC 1 for the spectral library from 204 vials of chlorothiazide sodium. Important peaks are marked with arrows.

    Article Snippet: For instance, Mylan, one of the manufacturers, discontinued its chlorothiazide injection in May 2020.

    Techniques: