nkcc1 blocker bumetanide  (Tocris)

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

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

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( A ) Log 2 -normalised expression of Th, Drd1, Drd2, Drd3, Drd4, and Drd5 mRNA in olfactory bulb (OB) DA cells. ( B ) Example trace of an action potential fired by a putative anaxonic DA neuron (left) and its monophasic phase-plane plot profile (right). Note the prolonged repolarisation due to Cs-based internal solution. ( C ) Example traces of an auto-evoked inhibition (AEI) response recorded before (magenta) and after (grey) the application of gabazine. The subtraction is shown in the orange inset trace. ( D ) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated by dopamine released from the patched DA cell. ( E ) Example traces of an AEI response before (purple) and after (green) applying D1-like and D2-like receptor blockers (SR 95531 hydrobromide and sulpiride, each at 10 µM). ( F ) AEI charge before (purple) and after (green) applying dopamine receptor antagonists; n=6 cells from N=4 mice; paired t-test, p=0.21, n.s.=non-significant. ( G ) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated via gap junctions. ( H ) Example trace of an AEI response in the presence of the gap junction blocker carbenoxolone at 100 µM. ( I ) AEI charge in the presence of carbenoxolone at 100 µM. Each dot shows one cell; lines show mean ± SEM; n=9 cells from N=4 mice. ( J ) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated by depolarising GABA released from the patched cell. ( K ) Example trace of an AEI response in the presence of the NKCC1 blocker bumetanide at 20 µM. ( L ) AEI charge in the presence of bumetanide at 20 µM. All conventions as in F ; n=5 cells from N=3 mice.

Article Snippet: The gap junction blocker carbenoxolone disodium at 100 μM (3096, Tocris) and the NKCC1 blocker bumetanide at 20 μM (3108, Tocris) were applied in the bath from the beginning and kept in the ACSF for at least 20–40 min. We adopted this strategy due to two key considerations: (1) carbenoxolone-induced instability in the membrane properties of the neurons during the initial minutes of application, and (2) sufficient time was required for chloride transporters to reach a steady intracellular chloride concentration ([Cl - ]) in response to bumetanide.

Techniques: Expressing, Inhibition

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( A ) Log 2 -normalised expression of Th, Drd1, Drd2, Drd3, Drd4, and Drd5 mRNA in olfactory bulb (OB) DA cells. ( B ) Example trace of an action potential fired by a putative anaxonic DA neuron (left) and its monophasic phase-plane plot profile (right). Note the prolonged repolarisation due to Cs-based internal solution. ( C ) Example traces of an auto-evoked inhibition (AEI) response recorded before (magenta) and after (grey) the application of gabazine. The subtraction is shown in the orange inset trace. ( D ) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated by dopamine released from the patched DA cell. ( E ) Example traces of an AEI response before (purple) and after (green) applying D1-like and D2-like receptor blockers (SR 95531 hydrobromide and sulpiride, each at 10 µM). ( F ) AEI charge before (purple) and after (green) applying dopamine receptor antagonists; n=6 cells from N=4 mice; paired t-test, p=0.21, n.s.=non-significant. ( G ) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated via gap junctions. ( H ) Example trace of an AEI response in the presence of the gap junction blocker carbenoxolone at 100 µM. ( I ) AEI charge in the presence of carbenoxolone at 100 µM. Each dot shows one cell; lines show mean ± SEM; n=9 cells from N=4 mice. ( J ) Schematic showing the potential involvement in the AEI response of neighbouring GABAergic neurons activated by depolarising GABA released from the patched cell. ( K ) Example trace of an AEI response in the presence of the NKCC1 blocker bumetanide at 20 µM. ( L ) AEI charge in the presence of bumetanide at 20 µM. All conventions as in F ; n=5 cells from N=3 mice.

Article Snippet: Chemical compound, drug , Bumetanide , Tocris , Catalogue number 3108 , Used at 20 μM.

Techniques: Expressing, Inhibition