Journal: Journal of cellular and molecular medicine

Article Title: Elucidation of Dexmedetomidine-Induced Analgesic Tolerance Mechanisms in Neuropathic Pain With Modulation of SGK1, NR2A, and NR2B Expression via the Spinal SGK1/NF-κB Signalling Pathway.

doi: 10.1111/jcmm.70372

Figure Lengend Snippet: FIGURE 5 | Inhibition of SGK1 Pathway antagonises development of DEX analgesic tolerance (A) Schematic diagram of the experimental pro- cedure. (B–F) represented the PWT (contralateral and ipsilateral), the PWL, TFL, and WBR of the mice in different treatment groups, respective- ly. SNI+ Vehicle: SNI group; SNI + m-GSK650394: Multiple GSK650394 injections; SNI + s-DEX + vehicle: Single DEX injection; SNI + s-DEX + s- GSK650394: Single DEX injection + single GSK650394 injection; SNI + m-DEX + Vehicle: Multiple DEX injections; SNI + m-DEX + m-GSK650394: Multiple DEX + multiple GSK650394 injections. (G) Western blotted bands of SGK1, pSGK1 with GAPDH as an internal control. (H and I) relative protein levels of SGK1 and pSGK1. (J) Western blotted bands of NR1, NR2A, and NR2B with GAPDH as an internal control. (L–N) relative protein levels of NR1, NR2A and NR2B. Data were expressed as mean ± SD. Different letters indicated significant differences among the groups at (p < 0.05). *p < 0.05 indicates that the measured value at this measurement time point was significantly different from that of the SNI group, **p < 0.01.

Article Snippet: Drug or antibody name Company name Dexmedetomidine Hangzhou Aladdin Biotechnology Co. Ltd. PEG300 Medical Chemicals & Engineering (MCE) PDTC inhibitor Sigma- Aldrich Inc. (Saint Louis, MO, USA) GSK650394 inhibitor Medical Chemicals & Engineering (MCE) NR1 antibody (1:500) Abclonal Technology Co. Ltd. NR2A antibody (1:500) Abclonal Technology Co. Ltd. NR2B antibody (1:500) Abclonal Technology Co. Ltd. SGK1 (1:500) Abclonal Technology Co. Ltd. pSGK1(Bioss, diluted 1:200) Bioss Biotechnology Co. Ltd. p65 Antibody (1:500) Abclonal Technology Co. Ltd. p- p65 Antibody (Abclonal, diluted 1:200) Abclonal Technology Co. Ltd. GAPDH Antibody (1:1000) Abclonal Technology Co. Ltd. Donkey anti- Rabbit IgG Secondary Antibody Abclonal Technology Co. Ltd. Cy3 Goat anti- Rabbit Secondary Antibody (1:400) Wuhan Cyvellon Biotech Co. Ltd. FITC Goat anti- Mouse Secondary Antibody (1:400) Wuhan Cyvellon Biotech Co. Ltd. NeuN Antibody (Proteintech, diluted 1:200) Proteintech Group Inc. GFAP Antibody (Proteintech, diluted 1:200) Proteintech Group Inc. 15824934, 2025, 6, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/jcm m .70372 by IN A SP - N E PA L , W iley O nline L ibrary on [21/03/2025].

Techniques: Inhibition, Injection, Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of SGK1 Alleviates Apoptosis and Oxidative Stress in Dry Eye Disease by Activating FoxO3a-Mediated Autophagy

doi: 10.1167/iovs.67.5.37

Figure Lengend Snippet: Inhibition of SGK1 attenuates apoptosis and oxidative stress in hyperosmotic-treated HCECs. ( A ) Quantitative analysis of cell viability ( n = 5). ( B ) Western blot analysis of SGK1, Bcl-2, Bax, and cCasp3. ( C ) Quantification of protein levels from B ( n = 3). ( D ) Flow cytometric analysis of apoptosis. ( E ) Apoptosis rate quantification ( n = 3). ( F ) Fluorescence images of intracellular ROS. Scale bar : 100 µm. ( G ) Flow cytometric measurement of ROS levels. ( H ) Quantitative analysis of ROS levels from G ( n = 3). ( I ) Measurement of MDA content ( n = 3). ( J ) CAT activity assay ( n = 3). ( K ) SOD activity assay ( n = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The GSK650394 group received 5 μL of 5-mM GSK650394 eye drops (TargetMol Chemicals, Boston, MA, USA) twice daily, the rapamycin (RAPA; Selleck Chemicals, Shanghai, China) group received 5 μL of 0.25% RAPA eye drops twice daily, the dimethyl sulfoxide (DMSO; TargetMol Chemicals) group received 5 μL of PBS containing 0.1% DMSO twice daily serving as a solvent control, and the 3-methyladenine (3-MA; Selleck Chemicals) group received 5 μL of 0.5% 3-MA eye drops twice daily in addition to GSK650394 treatment.

Techniques: Inhibition, Analysis, Western Blot, Fluorescence, Activity Assay, FACS, Fluorescence Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of SGK1 Alleviates Apoptosis and Oxidative Stress in Dry Eye Disease by Activating FoxO3a-Mediated Autophagy

doi: 10.1167/iovs.67.5.37

Figure Lengend Snippet: Inhibition of SGK1 alleviates ocular surface damage in DED mice. ( A ) Immunofluorescence staining of SGK1 ( red ) and DAPI ( blue ) in mouse corneas. Scale bar : 50 µm. ( B ) Tear secretion analysis ( n = 5). ( C ) Corneal fluorescein staining (days 0, 7, and 14). ( D ) Clinical score quantification corresponding to C ( n = 5). ( E ) H&E staining of corneas on day 14. Scale bar : 50 µm. Also shown is quantification of corneal epithelial thickness ( n = 5). ( F ) PAS staining of conjunctival sections on day 14. Scale bar : 50 µm. Also shown is quantification of goblet cell density ( n = 5). ( G ) TUNEL assay of apoptotic corneal and conjunctival epithelia ( red ) with DAPI ( blue ). Scale bar : 50 µm. Also shown is quantification of TUNEL-positive cells ( n = 5). All data are presented as mean ± SD. ** P < 0.01, *** P < 0.001.

Article Snippet: The GSK650394 group received 5 μL of 5-mM GSK650394 eye drops (TargetMol Chemicals, Boston, MA, USA) twice daily, the rapamycin (RAPA; Selleck Chemicals, Shanghai, China) group received 5 μL of 0.25% RAPA eye drops twice daily, the dimethyl sulfoxide (DMSO; TargetMol Chemicals) group received 5 μL of PBS containing 0.1% DMSO twice daily serving as a solvent control, and the 3-methyladenine (3-MA; Selleck Chemicals) group received 5 μL of 0.5% 3-MA eye drops twice daily in addition to GSK650394 treatment.

Techniques: Inhibition, Immunofluorescence, Staining, Analysis, TUNEL Assay, H&E Stain

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of SGK1 Alleviates Apoptosis and Oxidative Stress in Dry Eye Disease by Activating FoxO3a-Mediated Autophagy

doi: 10.1167/iovs.67.5.37

Figure Lengend Snippet: Inhibition of SGK1 promotes autophagy in DED. ( A ) Western blot analysis of p62 and LC3 in HCECs. ( B ) Quantification of protein levels from A ( n = 3). ( C ) Western blot analysis of p62 and LC3 in mouse corneal tissues ( n = 5 corneas per sample). ( D ) Quantification of protein levels from C ( n = 3). ( E ) Fluorescent images of HCECs transfected with mRFP-GFP-LC3 lentivirus, showing GFP-LC3 ( green ) and RFP-LC3 ( red ) signals. Scale bar : 10 µm. ( F ) Quantitative analysis of autolysosomes ( red puncta ) and autophagosomes ( yellow puncta ) from E ( n = 5). ( G ) TEM images of HCECs. Scale bar : 1 µm. A rrows indicate autophagic vacuoles, including autophagosomes and autolysosomes. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The GSK650394 group received 5 μL of 5-mM GSK650394 eye drops (TargetMol Chemicals, Boston, MA, USA) twice daily, the rapamycin (RAPA; Selleck Chemicals, Shanghai, China) group received 5 μL of 0.25% RAPA eye drops twice daily, the dimethyl sulfoxide (DMSO; TargetMol Chemicals) group received 5 μL of PBS containing 0.1% DMSO twice daily serving as a solvent control, and the 3-methyladenine (3-MA; Selleck Chemicals) group received 5 μL of 0.5% 3-MA eye drops twice daily in addition to GSK650394 treatment.

Techniques: Inhibition, Western Blot, Analysis, Transfection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of SGK1 Alleviates Apoptosis and Oxidative Stress in Dry Eye Disease by Activating FoxO3a-Mediated Autophagy

doi: 10.1167/iovs.67.5.37

Figure Lengend Snippet: Inhibition of SGK1 promotes autophagy through FoxO3a activation. ( A ) Co-IP and western blot analysis of SGK1 and FoxO3a in HCECs. ( B ) Western blot analysis of p -FoxO3a and FoxO3a in HCECs. ( C ) Quantification of protein levels from B ( n = 3). ( D ) Immunofluorescence staining of FoxO3a ( green ) and DAPI ( blue ) in HCECs. Scale bar : 5 µm. ( E ) Immunofluorescence staining of FoxO3a ( green ) and DAPI ( blue ) in mouse corneal epithelium. Scale bar : 50 µm. ( F ) Relative mRNA expression level of FoxO3a in HCECs ( n = 3). ( G ) Western blot analysis of FoxO3a protein in HCECs. ( H ) Quantification of FoxO3a protein levels from G ( n = 3). ( I ) Western blot analysis of FoxO3a, p62, and LC3 in HCECs. ( J ) Quantification of protein levels from I ( n = 3). ( K ) Fluorescent images of HCECs transfected with mRFP-GFP-LC3 lentivirus, showing GFP-LC3 ( green ) and RFP-LC3 ( red ) signals. Scale bar : 10 µm. ( L ) Quantitative analysis of autolysosomes ( red puncta ) and autophagosomes ( yellow puncta ) from K ( n = 5). ( M ) TEM images of HCECs. Scale bar : 1 µm. Arrows indicate autophagic vacuoles, including autophagosomes and autolysosomes. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The GSK650394 group received 5 μL of 5-mM GSK650394 eye drops (TargetMol Chemicals, Boston, MA, USA) twice daily, the rapamycin (RAPA; Selleck Chemicals, Shanghai, China) group received 5 μL of 0.25% RAPA eye drops twice daily, the dimethyl sulfoxide (DMSO; TargetMol Chemicals) group received 5 μL of PBS containing 0.1% DMSO twice daily serving as a solvent control, and the 3-methyladenine (3-MA; Selleck Chemicals) group received 5 μL of 0.5% 3-MA eye drops twice daily in addition to GSK650394 treatment.

Techniques: Inhibition, Activation Assay, Co-Immunoprecipitation Assay, Western Blot, Analysis, Immunofluorescence, Staining, Expressing, Transfection, Knockdown

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of SGK1 Alleviates Apoptosis and Oxidative Stress in Dry Eye Disease by Activating FoxO3a-Mediated Autophagy

doi: 10.1167/iovs.67.5.37

Figure Lengend Snippet: Schematic illustration of the mechanism by which inhibition of SGK1 alleviates apoptosis and oxidative stress in DED by activating FoxO3a-mediated autophagy. (Created with BioGDP.com . ).

Article Snippet: The GSK650394 group received 5 μL of 5-mM GSK650394 eye drops (TargetMol Chemicals, Boston, MA, USA) twice daily, the rapamycin (RAPA; Selleck Chemicals, Shanghai, China) group received 5 μL of 0.25% RAPA eye drops twice daily, the dimethyl sulfoxide (DMSO; TargetMol Chemicals) group received 5 μL of PBS containing 0.1% DMSO twice daily serving as a solvent control, and the 3-methyladenine (3-MA; Selleck Chemicals) group received 5 μL of 0.5% 3-MA eye drops twice daily in addition to GSK650394 treatment.

Techniques: Inhibition

Journal: European journal of cell biology

Article Title: SGK1 is essential for meiotic resumption in mammalian oocytes

doi: 10.1016/j.ejcb.2022.151210

Figure Lengend Snippet: Of the SGK genes only SGK1 is expressed in the mouse oocyte and its inhibition hinders nuclear envelope breakdown. A) WB analysis of the expression of the three SGK proteins in oocytes (30 or 100 per sample) and control tissue; GAPDH was used as loading control. The images are representative from at least three biological replicates. For mRNA expression see . B) Quantification of oocytes undergoing nuclear envelope breakdown (NEBD) in the control (0.06% vehicle, DMSO) and presence of SGK1 inhibitor (GSK-650394). Data are represented as the mean ± SEM of at least three independent experiments; n ≥ 44 oocytes; ns, not significant; *** p < 0.001; ** p < 0.01 according to One-way ANOVA after arcsine transformation. For inhibitor validation see . C) Timing of oocyte NEBD after IBMX wash in absence (Control, 0.06% vehicle DMSO) or presence of SGK1 inhibitor (0.01 mM) for 10 h and 1 h. Box plot displays mean, 25th and 75th percentile and ± SD of at least three independent experiments; n ≥ 45 oocytes; ns, not significant; ** p < 0.01 according One-way ANOVA. D) Timing of oocyte cytokinesis (polar body extrusion) in absence (control, 0.06% vehicle DMSO) or presence of SGK1 inhibitor (0.01 mM) added at different time points post-IBMX wash. Box plot displays mean, 25th and 75th percentile and ± SD of at least three independent experiments; n ≥ 26 oocytes; ns, not significant; * p < 0.05; *** p < 0.001; according One-way ANOVA.

Article Snippet: To unveil this role, we treated GV oocytes with a specific SGK1 inhibitor (GSK-650394, Merck, Darmstadt, Germany; ) which restricts SGK1 activity (and SGK2 with less affinity) and has been already used in several fields of research ( ; ; ).

Techniques: Inhibition, Expressing, Control, Transformation Assay, Biomarker Discovery

Journal: European journal of cell biology

Article Title: SGK1 is essential for meiotic resumption in mammalian oocytes

doi: 10.1016/j.ejcb.2022.151210

Figure Lengend Snippet: Active SGK1 is concentrated in the oocyte nucleus and its expression decreases along the first meiotic division. A) Immunocytochemistry shows dominant localization of SGK1 phosphorylated at Thr256 in the oocyte nucleus (grey and red). DAPI (blue), scale bar 15 μm. B) Quantification of SGK1 (Thr256) fluorescence at different oocyte areas and stages of meiosis. Data are represented as the mean ± SEM of at least three independent experiments normalized to the group with highest intensity (GV) as 100% fluorescence; n ≥ 40 oocytes; ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 according to One-way ANOVA for comparing oocyte stages and Student’s t test for comparing oocyte areas.

Article Snippet: To unveil this role, we treated GV oocytes with a specific SGK1 inhibitor (GSK-650394, Merck, Darmstadt, Germany; ) which restricts SGK1 activity (and SGK2 with less affinity) and has been already used in several fields of research ( ; ; ).

Techniques: Expressing, Immunocytochemistry, Fluorescence

Journal: European journal of cell biology

Article Title: SGK1 is essential for meiotic resumption in mammalian oocytes

doi: 10.1016/j.ejcb.2022.151210

Figure Lengend Snippet: Inhibition of SGK1 impairs CDK1 activation through CDC25B in the oocyte prior to NEBD. A) WB analysis of CDK1 (Tyr 15) at different timing of oocyte meiotic resumption in absence (control, 0.02% vehicle DMSO) and presence of SGK1 inhibitor (GSK-650394; 0.01 mM). GAPDH and CDK1 (total) were used as a loading control. The images are representative from at least three biological replicates of 30 oocytes per sample. B) WB quantification of CDK1 (Tyr15) normalized to CDK1 (total). Data are represented as the mean ± SEM from at least three independent experiments; n = 30 oocytes per sample; ns, not significant, * p < 0.05, *** p < 0.001 according to Student’s t test. C) WB analysis of CDC25B at different timing of oocyte meiotic resumption in absence (control, 0.02% vehicle DMSO) and presence of SGK1 inhibitor (0.01 mM). GAPDH was used as a loading control. The images are representative from at least three biological replicates of 30 oocytes per sample. The arrowheads depict phosphorylated variants of CDC25B. D) WB quantification of CDC25B protein normalized to GAPDH. Data are represented as the mean ± SEM from at least three independent experiments; 30 oocytes per sample; ns = not significant, * p < 0.05, *** p < 0.001 according to Student’s t test.

Article Snippet: To unveil this role, we treated GV oocytes with a specific SGK1 inhibitor (GSK-650394, Merck, Darmstadt, Germany; ) which restricts SGK1 activity (and SGK2 with less affinity) and has been already used in several fields of research ( ; ; ).

Techniques: Inhibition, Activation Assay, Control

Journal: European journal of cell biology

Article Title: SGK1 is essential for meiotic resumption in mammalian oocytes

doi: 10.1016/j.ejcb.2022.151210

Figure Lengend Snippet: The phenotype resulting from SGK1 inhibition can be reversed by activation of CDK1. A) Quantification of oocytes undergoing NEBD after microinjection with RNA coding for H2b-gfp RNA (control) in the presence of SGK1 inhibitor or microinjected with RNA coding for H2B-GFP + CDK1-AF RNA in the presence of SGK1 inhibitor or IBMX. Data are represented as mean ± SEM of at least three independent experiments; n = 39 oocytes per group; ns, not significant, * p < 0.05, *** p < 0.001 according to One-way ANOVA. B) Timing of NEBD after IBMX wash in oocytes microinjected with H2B-GFP RNA (control) in the presence of inhibitor or microinjected with H2B-GFP+CDK1-AF RNA in absence and presence of SGK1 inhibitor. Box plot displays mean, 25th and 75th percentile and ± SD of at least three independent experiments; n ≥ 30 oocytes per group; ns, not significant; *** p < 0.001 according to One-way ANOVA.

Article Snippet: To unveil this role, we treated GV oocytes with a specific SGK1 inhibitor (GSK-650394, Merck, Darmstadt, Germany; ) which restricts SGK1 activity (and SGK2 with less affinity) and has been already used in several fields of research ( ; ; ).

Techniques: Inhibition, Activation Assay, Microinjection, Control

Journal: European journal of cell biology

Article Title: SGK1 is essential for meiotic resumption in mammalian oocytes

doi: 10.1016/j.ejcb.2022.151210

Figure Lengend Snippet: Hypothesis of role of the SGK1 in the resumption of meiosis. At the GV stage, SGK1 (Thr256) is active enclosed in the oocyte nucleus without effect on meiotic resumption. PKA is active when cAMP levels are high, phosphorylating CDC25B (Ser321), inhibiting this phosphatase and keeping it in the cytoplasm. Prior to nuclear envelope breakdown (NEBD), cAMP levels decline, PKA becomes inactive and CDC25B is dephosphorylated. Consequently, CDC25B localize to the nucleus where active SGK1 (Thr256) phosphorylate the activation sites of CDC25B, and thus promote the NEBD process.

Article Snippet: To unveil this role, we treated GV oocytes with a specific SGK1 inhibitor (GSK-650394, Merck, Darmstadt, Germany; ) which restricts SGK1 activity (and SGK2 with less affinity) and has been already used in several fields of research ( ; ; ).

Techniques: Activation Assay