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  • 92
    Proteintech anti stbd1
    ER stress activation induces glycogenesis and the formation of glycogen-containing ER structures. (A) Representative immunofluorescence images of C2C12 myoblasts treated with TM or DMSO (control) and stained for glycogen and <t>Stbd1.</t> TM-treated cells display prominent glycogen structures that strongly coincide with Stbd1 (overlay) [mean±s.e.m. Manders’ colocalization coefficient (MCC), 0.746±0.032; n =10]. DMSO-treated controls occasionally display smaller Stbd1-positive glycogen structures (arrowheads) (MCC, 0.615±0.025; n =6). (B) Glycogen quantification in DMSO- and TM-treated C2C12 myoblasts revealed that ER stress activation stimulates glycogen synthesis (mean±s.e.m. μg glucose/mg protein: DMSO-treated, 0.55±0.20; TM-treated, 9.56±0.74; n =3). *** P ≤0.001 (one-tailed unpaired Student's t -test). (C) Representative images of TM- and DMSO-treated C2C12 myoblasts immunostained with the indicated antibodies. In addition to positive Stbd1 immunostaining, both the ER stress-induced glycogen structures and those sporadically present in DMSO-treated controls (arrowheads) stain positive for GS1 (MCC: TM-treated, 0.785±0.012, n =10; DMSO-treated, 0.565±0.052, n =7), GN (MCC: TM-treated, 0.791±0.011, n =10; DMSO-treated, 0.703±0.025, n =8) as well as calnexin (MCC: TM-treated, 0.752±0.019, n =10; DMSO-treated, 0.763±0.040, n =7) but not Lamp1 (MCC: TM-treated, 0.047±0.010, n =10; DMSO-treated, 0.028±0.009, n =10). Inserts show single stainings at higher magnification for the corresponding areas indicated by dashed boxes. (D–F) Transmission electron micrographs of TM- (D,E) and DMSO- treated (F) C2C12 cells. TM-treated cells display prominent membrane-free glycogen structures not present in DMSO-treated control cells. A higher magnification image of the boxed area in D is shown in E. Scale bars: 20 μm (A,C), 2 μm (D,F), 1 μm (E).
    Anti Stbd1, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti stbd1/product/Proteintech
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti stbd1 - by Bioz Stars, 2024-03
    92/100 stars
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    92
    Cayman Chemical 10 gingerol
    Human neutrophils were isolated from healthy volunteers and then treated with various stimuli for 3 hours in the presence of different gingerol analogues. NETosis was quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). Dose response to LPS-mediated NETosis upon treatment with 6-gingerol ( A ), 8-gingerol ( B ), and <t>10-gingerol</t> ( C ). NETosis in response to PMA ( D ), RNP ICs ( E ), and APS IgG ( F ) was quantified in the presence of 10 μM gingerol. NETosis was assessed by immunofluorescence microscopy ( G ). Neutrophils were treated with LPS, PMA, RNP ICs, or APS IgG in the presence or absence of 6-gingerol (10 μM). Blue, DNA; green, extracellular neutrophil elastase. Scale bar: 100 microns. For A – F , mean and SEM are presented for n = 3 independent experiments; * P < 0.05, ** P < 0.01, **** P < 0.0001 as compared with the 0 μM gingerol group by 1-way ANOVA corrected with Dunnett’s test.
    10 Gingerol, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/10 gingerol/product/Cayman Chemical
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    10 gingerol - by Bioz Stars, 2024-03
    92/100 stars
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    92
    Proteintech stbd1
    Human neutrophils were isolated from healthy volunteers and then treated with various stimuli for 3 hours in the presence of different gingerol analogues. NETosis was quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). Dose response to LPS-mediated NETosis upon treatment with 6-gingerol ( A ), 8-gingerol ( B ), and <t>10-gingerol</t> ( C ). NETosis in response to PMA ( D ), RNP ICs ( E ), and APS IgG ( F ) was quantified in the presence of 10 μM gingerol. NETosis was assessed by immunofluorescence microscopy ( G ). Neutrophils were treated with LPS, PMA, RNP ICs, or APS IgG in the presence or absence of 6-gingerol (10 μM). Blue, DNA; green, extracellular neutrophil elastase. Scale bar: 100 microns. For A – F , mean and SEM are presented for n = 3 independent experiments; * P < 0.05, ** P < 0.01, **** P < 0.0001 as compared with the 0 μM gingerol group by 1-way ANOVA corrected with Dunnett’s test.
    Stbd1, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/stbd1/product/Proteintech
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    stbd1 - by Bioz Stars, 2024-03
    92/100 stars
      Buy from Supplier

    86
    NCIMB Ltd strain ncimb 11842
    Human neutrophils were isolated from healthy volunteers and then treated with various stimuli for 3 hours in the presence of different gingerol analogues. NETosis was quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). Dose response to LPS-mediated NETosis upon treatment with 6-gingerol ( A ), 8-gingerol ( B ), and <t>10-gingerol</t> ( C ). NETosis in response to PMA ( D ), RNP ICs ( E ), and APS IgG ( F ) was quantified in the presence of 10 μM gingerol. NETosis was assessed by immunofluorescence microscopy ( G ). Neutrophils were treated with LPS, PMA, RNP ICs, or APS IgG in the presence or absence of 6-gingerol (10 μM). Blue, DNA; green, extracellular neutrophil elastase. Scale bar: 100 microns. For A – F , mean and SEM are presented for n = 3 independent experiments; * P < 0.05, ** P < 0.01, **** P < 0.0001 as compared with the 0 μM gingerol group by 1-way ANOVA corrected with Dunnett’s test.
    Strain Ncimb 11842, supplied by NCIMB Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/strain ncimb 11842/product/NCIMB Ltd
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    strain ncimb 11842 - by Bioz Stars, 2024-03
    86/100 stars
      Buy from Supplier

    Image Search Results


    ER stress activation induces glycogenesis and the formation of glycogen-containing ER structures. (A) Representative immunofluorescence images of C2C12 myoblasts treated with TM or DMSO (control) and stained for glycogen and Stbd1. TM-treated cells display prominent glycogen structures that strongly coincide with Stbd1 (overlay) [mean±s.e.m. Manders’ colocalization coefficient (MCC), 0.746±0.032; n =10]. DMSO-treated controls occasionally display smaller Stbd1-positive glycogen structures (arrowheads) (MCC, 0.615±0.025; n =6). (B) Glycogen quantification in DMSO- and TM-treated C2C12 myoblasts revealed that ER stress activation stimulates glycogen synthesis (mean±s.e.m. μg glucose/mg protein: DMSO-treated, 0.55±0.20; TM-treated, 9.56±0.74; n =3). *** P ≤0.001 (one-tailed unpaired Student's t -test). (C) Representative images of TM- and DMSO-treated C2C12 myoblasts immunostained with the indicated antibodies. In addition to positive Stbd1 immunostaining, both the ER stress-induced glycogen structures and those sporadically present in DMSO-treated controls (arrowheads) stain positive for GS1 (MCC: TM-treated, 0.785±0.012, n =10; DMSO-treated, 0.565±0.052, n =7), GN (MCC: TM-treated, 0.791±0.011, n =10; DMSO-treated, 0.703±0.025, n =8) as well as calnexin (MCC: TM-treated, 0.752±0.019, n =10; DMSO-treated, 0.763±0.040, n =7) but not Lamp1 (MCC: TM-treated, 0.047±0.010, n =10; DMSO-treated, 0.028±0.009, n =10). Inserts show single stainings at higher magnification for the corresponding areas indicated by dashed boxes. (D–F) Transmission electron micrographs of TM- (D,E) and DMSO- treated (F) C2C12 cells. TM-treated cells display prominent membrane-free glycogen structures not present in DMSO-treated control cells. A higher magnification image of the boxed area in D is shown in E. Scale bars: 20 μm (A,C), 2 μm (D,F), 1 μm (E).

    Journal: Journal of Cell Science

    Article Title: Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts

    doi: 10.1242/jcs.244855

    Figure Lengend Snippet: ER stress activation induces glycogenesis and the formation of glycogen-containing ER structures. (A) Representative immunofluorescence images of C2C12 myoblasts treated with TM or DMSO (control) and stained for glycogen and Stbd1. TM-treated cells display prominent glycogen structures that strongly coincide with Stbd1 (overlay) [mean±s.e.m. Manders’ colocalization coefficient (MCC), 0.746±0.032; n =10]. DMSO-treated controls occasionally display smaller Stbd1-positive glycogen structures (arrowheads) (MCC, 0.615±0.025; n =6). (B) Glycogen quantification in DMSO- and TM-treated C2C12 myoblasts revealed that ER stress activation stimulates glycogen synthesis (mean±s.e.m. μg glucose/mg protein: DMSO-treated, 0.55±0.20; TM-treated, 9.56±0.74; n =3). *** P ≤0.001 (one-tailed unpaired Student's t -test). (C) Representative images of TM- and DMSO-treated C2C12 myoblasts immunostained with the indicated antibodies. In addition to positive Stbd1 immunostaining, both the ER stress-induced glycogen structures and those sporadically present in DMSO-treated controls (arrowheads) stain positive for GS1 (MCC: TM-treated, 0.785±0.012, n =10; DMSO-treated, 0.565±0.052, n =7), GN (MCC: TM-treated, 0.791±0.011, n =10; DMSO-treated, 0.703±0.025, n =8) as well as calnexin (MCC: TM-treated, 0.752±0.019, n =10; DMSO-treated, 0.763±0.040, n =7) but not Lamp1 (MCC: TM-treated, 0.047±0.010, n =10; DMSO-treated, 0.028±0.009, n =10). Inserts show single stainings at higher magnification for the corresponding areas indicated by dashed boxes. (D–F) Transmission electron micrographs of TM- (D,E) and DMSO- treated (F) C2C12 cells. TM-treated cells display prominent membrane-free glycogen structures not present in DMSO-treated control cells. A higher magnification image of the boxed area in D is shown in E. Scale bars: 20 μm (A,C), 2 μm (D,F), 1 μm (E).

    Article Snippet: The following primary antibodies were used in this study: anti-Stbd1 (11842-1-AP; Proteintech; 1:500), anti-calnexin (ADI-SPA-860-D; Enzo Life Sciences; 1:250), anti-Lamp1 (1D4B; Developmental Studies Hybridoma Bank; 1:50) and anti-LC3 (NB100-2220; Novus Biologicals; 1:435).

    Techniques: Activation Assay, Immunofluorescence, Staining, One-tailed Test, Immunostaining, Transmission Assay

    ER stress-induced structures contain α-amylase-degradable glycogen. TM-treated C2C12 cells were incubated either in the absence (A) or presence (B) of α-amylase and double-stained for Stbd1 (green) and glycogen, GS1 or GN (red). α-amylase treatment resulted in glycogen depletion from the ER stress-induced structures, which still displayed positive immunostaining for Stbd1 [mean±s.e.m. Manders’ colocalization coefficient (MCC): −α-amylase, 0.675±0.008, n =10; +α-amylase, 0.022±0.006, n =10], GS1 (MCC: 0.661±0.031, n =10) and GN (MCC: 0.528±0.025, n =10). Overlays are shown as inserts and represent higher magnifications of the indicated boxed areas. Representative images are shown. Scale bar: 20 μm.

    Journal: Journal of Cell Science

    Article Title: Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts

    doi: 10.1242/jcs.244855

    Figure Lengend Snippet: ER stress-induced structures contain α-amylase-degradable glycogen. TM-treated C2C12 cells were incubated either in the absence (A) or presence (B) of α-amylase and double-stained for Stbd1 (green) and glycogen, GS1 or GN (red). α-amylase treatment resulted in glycogen depletion from the ER stress-induced structures, which still displayed positive immunostaining for Stbd1 [mean±s.e.m. Manders’ colocalization coefficient (MCC): −α-amylase, 0.675±0.008, n =10; +α-amylase, 0.022±0.006, n =10], GS1 (MCC: 0.661±0.031, n =10) and GN (MCC: 0.528±0.025, n =10). Overlays are shown as inserts and represent higher magnifications of the indicated boxed areas. Representative images are shown. Scale bar: 20 μm.

    Article Snippet: The following primary antibodies were used in this study: anti-Stbd1 (11842-1-AP; Proteintech; 1:500), anti-calnexin (ADI-SPA-860-D; Enzo Life Sciences; 1:250), anti-Lamp1 (1D4B; Developmental Studies Hybridoma Bank; 1:50) and anti-LC3 (NB100-2220; Novus Biologicals; 1:435).

    Techniques: Incubation, Staining, Immunostaining

    Stbd1 is upregulated in response to ER stress and is required for the formation of glycogen structures. (A) Western blot (top) and densitometry (bottom; mean±s.e.m., n =3) of protein extracts from C2C12 myoblasts collected after 0, 4, 8 or 16 h of TM treatment, showing prominent upregulation of Stbd1. Induction of ER stress was verified by the increase in BiP protein levels, and Gapdh was used as a loading control. (B) Western immunoblot (top) and densitometry (bottom; mean±s.e.m., n =3) for the assessment of Stbd1 silencing efficiency. Protein extracts from non-transduced controls (C2C12) and C2C12 myoblasts expressing either a scrambled (shScr) or a Stbd1-specific (shStbd1) shRNA sequence were prepared in the absence (−) or presence of TM treatment (+) and probed for Stbd1. Non-transduced and shScr cells display increased Stbd1 protein levels following ER stress activation, in contrast to shStbd1 cells. BiP was employed to evaluate ER stress activation, and Gapdh was used as a loading control. (C) Representative images of shScr and shStbd1 cells immunostained for glycogen after TM treatment. Stbd1 silencing impairs the formation of ER stress-induced glycogen clusters. (D) Quantification of glycogen levels in controls (non-transduced and shScr) and shStbd1 cells after treatment with DMSO (−) or TM (+), revealed no statistically significant difference (mean±s.e.m. μg glucose/mg protein: DMSO C2C12, 0.24±0.07; TM C2C12, 6.55±1.05; DMSO shScr, 0.23±0.11; TM shScr, 7.01±0.92; DMSO shStbd1, 0.17±0.08; TM shStbd1, 4.62±1.16; n =3). (E) Representative images of sh3′UTR C2C12 cells transiently transfected with the indicated vectors and immunostained for Myc and glycogen. Glycogen clustering is restored in cells transiently overexpressing Stbd1–Myc [mean±s.e.m. Manders’ colocalization coefficient (MCC): 0.800±0.026, n =10] or the W188A/V191A–Myc AIM mutant (MCC: 0.716±0.033, n =10) but not W273G–Myc (MCC: 0.051±0.014, n =10). Inserts show single stainings of the corresponding boxed areas at higher magnification. * P ≤0.05; ** P ≤0.01; ns, not significant (one-tailed unpaired Student's t -test). Scale bars: 20 μm.

    Journal: Journal of Cell Science

    Article Title: Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts

    doi: 10.1242/jcs.244855

    Figure Lengend Snippet: Stbd1 is upregulated in response to ER stress and is required for the formation of glycogen structures. (A) Western blot (top) and densitometry (bottom; mean±s.e.m., n =3) of protein extracts from C2C12 myoblasts collected after 0, 4, 8 or 16 h of TM treatment, showing prominent upregulation of Stbd1. Induction of ER stress was verified by the increase in BiP protein levels, and Gapdh was used as a loading control. (B) Western immunoblot (top) and densitometry (bottom; mean±s.e.m., n =3) for the assessment of Stbd1 silencing efficiency. Protein extracts from non-transduced controls (C2C12) and C2C12 myoblasts expressing either a scrambled (shScr) or a Stbd1-specific (shStbd1) shRNA sequence were prepared in the absence (−) or presence of TM treatment (+) and probed for Stbd1. Non-transduced and shScr cells display increased Stbd1 protein levels following ER stress activation, in contrast to shStbd1 cells. BiP was employed to evaluate ER stress activation, and Gapdh was used as a loading control. (C) Representative images of shScr and shStbd1 cells immunostained for glycogen after TM treatment. Stbd1 silencing impairs the formation of ER stress-induced glycogen clusters. (D) Quantification of glycogen levels in controls (non-transduced and shScr) and shStbd1 cells after treatment with DMSO (−) or TM (+), revealed no statistically significant difference (mean±s.e.m. μg glucose/mg protein: DMSO C2C12, 0.24±0.07; TM C2C12, 6.55±1.05; DMSO shScr, 0.23±0.11; TM shScr, 7.01±0.92; DMSO shStbd1, 0.17±0.08; TM shStbd1, 4.62±1.16; n =3). (E) Representative images of sh3′UTR C2C12 cells transiently transfected with the indicated vectors and immunostained for Myc and glycogen. Glycogen clustering is restored in cells transiently overexpressing Stbd1–Myc [mean±s.e.m. Manders’ colocalization coefficient (MCC): 0.800±0.026, n =10] or the W188A/V191A–Myc AIM mutant (MCC: 0.716±0.033, n =10) but not W273G–Myc (MCC: 0.051±0.014, n =10). Inserts show single stainings of the corresponding boxed areas at higher magnification. * P ≤0.05; ** P ≤0.01; ns, not significant (one-tailed unpaired Student's t -test). Scale bars: 20 μm.

    Article Snippet: The following primary antibodies were used in this study: anti-Stbd1 (11842-1-AP; Proteintech; 1:500), anti-calnexin (ADI-SPA-860-D; Enzo Life Sciences; 1:250), anti-Lamp1 (1D4B; Developmental Studies Hybridoma Bank; 1:50) and anti-LC3 (NB100-2220; Novus Biologicals; 1:435).

    Techniques: Western Blot, Expressing, shRNA, Sequencing, Activation Assay, Transfection, Mutagenesis, One-tailed Test

    ER stress-induced Stbd1 upregulation and glycogen clustering depends on PERK signalling. C2C12 cells were either left untreated (−) or cultured with TM (+) in the absence or presence of inhibitors of the individual UPR branches. (A) mRNA expression levels of representative UPR marker genes as assessed by qPCR. Data represent mean±s.e.m. of three independent experiments each performed in triplicate. (B) Representative images of double immunostaining for Stbd1 and glycogen revealed the lack of ER stress-induced, Stbd1-positive glycogen structures in the presence of the PERK inhibitor GSK2606414 [mean±s.e.m. Manders’ colocalization coefficient (MCC): no inhibitor, 0.870±0.011, n =10; 4μ8C, 0.768±0.021, n =10; AEBSF, 0.721±0.019, n =10]. Single stainings are shown as inserts representing higher magnifications of the corresponding boxed areas. (C) Western blot (top) and densitometry (bottom; mean±s.e.m., n =3) of protein extracts from cells treated as described above confirms the lack of ER stress-induced Stbd1 upregulation in the presence of the selective PERK inhibitor and a partial inhibition with AEBSF. Gapdh is shown as a loading control. * P ≤0.05; ** P ≤0.01; ns, not significant (one-tailed unpaired Student's t -test). Scale bar: 20 μm.

    Journal: Journal of Cell Science

    Article Title: Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts

    doi: 10.1242/jcs.244855

    Figure Lengend Snippet: ER stress-induced Stbd1 upregulation and glycogen clustering depends on PERK signalling. C2C12 cells were either left untreated (−) or cultured with TM (+) in the absence or presence of inhibitors of the individual UPR branches. (A) mRNA expression levels of representative UPR marker genes as assessed by qPCR. Data represent mean±s.e.m. of three independent experiments each performed in triplicate. (B) Representative images of double immunostaining for Stbd1 and glycogen revealed the lack of ER stress-induced, Stbd1-positive glycogen structures in the presence of the PERK inhibitor GSK2606414 [mean±s.e.m. Manders’ colocalization coefficient (MCC): no inhibitor, 0.870±0.011, n =10; 4μ8C, 0.768±0.021, n =10; AEBSF, 0.721±0.019, n =10]. Single stainings are shown as inserts representing higher magnifications of the corresponding boxed areas. (C) Western blot (top) and densitometry (bottom; mean±s.e.m., n =3) of protein extracts from cells treated as described above confirms the lack of ER stress-induced Stbd1 upregulation in the presence of the selective PERK inhibitor and a partial inhibition with AEBSF. Gapdh is shown as a loading control. * P ≤0.05; ** P ≤0.01; ns, not significant (one-tailed unpaired Student's t -test). Scale bar: 20 μm.

    Article Snippet: The following primary antibodies were used in this study: anti-Stbd1 (11842-1-AP; Proteintech; 1:500), anti-calnexin (ADI-SPA-860-D; Enzo Life Sciences; 1:250), anti-Lamp1 (1D4B; Developmental Studies Hybridoma Bank; 1:50) and anti-LC3 (NB100-2220; Novus Biologicals; 1:435).

    Techniques: Cell Culture, Expressing, Marker, Double Immunostaining, Western Blot, Inhibition, One-tailed Test

    Stbd1 overexpression is sufficient to induce glycogen clustering in the absence of ER stress but does not stimulate glycogenesis. (A) Representative images of C2C12 myoblasts stably overexpressing mouse Stbd1 (C2C12/Stbd1) or GFP as control (C2C12/GFP), stained using the indicated antibodies. C2C12/Stbd1 cells display large glycogen clusters that resemble those induced by ER stress and stain positive for Stbd1 [mean±s.e.m. Manders’ colocalization coefficient (MCC): 0.796±0.014, n =10], GS1 (MCC: 0.792±0.019, n =10), GN (MCC: 0.745±0.014, n =10), calnexin (MCC: 0.774±0.013, n =10) but not Lamp1 (MCC: 0.089±0.005, n =10). C2C12/GFP controls sporadically display smaller structures (arrowheads), with similar immunofluorescence staining for the above markers (MCC: Stbd1, 0.570±0.044, n =10; GS1, 0.618±0.021, n =8; GN, 0.518±0.033, n =8; calnexin, 0.646±0.053, n =5; Lamp1, 0.035±0.015, n =7). Inserts show single stainings of the corresponding boxed areas at higher magnification. (B) Western blot (top) and densitometry (bottom; mean±s.e.m., n =3) of protein lysates from C2C12/GFP and C2C12/Stbd1 cells confirms Stbd1 overexpression and the absence of ER stress activation in C2C12/Stbd1 cells, as assessed by BiP protein levels. Gapdh is shown as a loading control. (C) Quantification of intracellular glycogen levels in non-transduced C2C12, C2C12/GFP and C2C12/Stbd1 cells. Glycogen content in Stbd1-overexpressing cells is not statistically different from that of controls (mean±s.e.m, μg glucose/mg protein: C2C12, 1.56±0.27; C2C12/GFP, 1.87±0.30; C2C12/Stbd1, 2.27±0.60; n =3). *** P ≤0.001; ns, not significant (one-tailed unpaired Student's t -test). Scale bar: 20 μm.

    Journal: Journal of Cell Science

    Article Title: Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts

    doi: 10.1242/jcs.244855

    Figure Lengend Snippet: Stbd1 overexpression is sufficient to induce glycogen clustering in the absence of ER stress but does not stimulate glycogenesis. (A) Representative images of C2C12 myoblasts stably overexpressing mouse Stbd1 (C2C12/Stbd1) or GFP as control (C2C12/GFP), stained using the indicated antibodies. C2C12/Stbd1 cells display large glycogen clusters that resemble those induced by ER stress and stain positive for Stbd1 [mean±s.e.m. Manders’ colocalization coefficient (MCC): 0.796±0.014, n =10], GS1 (MCC: 0.792±0.019, n =10), GN (MCC: 0.745±0.014, n =10), calnexin (MCC: 0.774±0.013, n =10) but not Lamp1 (MCC: 0.089±0.005, n =10). C2C12/GFP controls sporadically display smaller structures (arrowheads), with similar immunofluorescence staining for the above markers (MCC: Stbd1, 0.570±0.044, n =10; GS1, 0.618±0.021, n =8; GN, 0.518±0.033, n =8; calnexin, 0.646±0.053, n =5; Lamp1, 0.035±0.015, n =7). Inserts show single stainings of the corresponding boxed areas at higher magnification. (B) Western blot (top) and densitometry (bottom; mean±s.e.m., n =3) of protein lysates from C2C12/GFP and C2C12/Stbd1 cells confirms Stbd1 overexpression and the absence of ER stress activation in C2C12/Stbd1 cells, as assessed by BiP protein levels. Gapdh is shown as a loading control. (C) Quantification of intracellular glycogen levels in non-transduced C2C12, C2C12/GFP and C2C12/Stbd1 cells. Glycogen content in Stbd1-overexpressing cells is not statistically different from that of controls (mean±s.e.m, μg glucose/mg protein: C2C12, 1.56±0.27; C2C12/GFP, 1.87±0.30; C2C12/Stbd1, 2.27±0.60; n =3). *** P ≤0.001; ns, not significant (one-tailed unpaired Student's t -test). Scale bar: 20 μm.

    Article Snippet: The following primary antibodies were used in this study: anti-Stbd1 (11842-1-AP; Proteintech; 1:500), anti-calnexin (ADI-SPA-860-D; Enzo Life Sciences; 1:250), anti-Lamp1 (1D4B; Developmental Studies Hybridoma Bank; 1:50) and anti-LC3 (NB100-2220; Novus Biologicals; 1:435).

    Techniques: Over Expression, Stable Transfection, Staining, Immunofluorescence, Western Blot, Activation Assay, One-tailed Test

    Stbd1 is not required for PTG-induced glycogen accumulation. (A) C2C12 myoblasts transiently transfected with PTG–Myc and immunostained with the indicated antibodies. PTG overexpression induces the build-up of glycogen clusters strongly colocalizing with PTG [mean±s.e.m. Manders’ colocalization coefficient (MCC): 0.775±0.023, n =10]. PTG-induced glycogen structures do not display any detectable Stbd1 (MCC: 0.024±0.006, n =10) or GS1 (MCC: 0.114±0.012, n =10) immunofluorescence; however, they stain weakly positive for GN (MCC: 0.624±0.013, n =10). No colocalization is observed with calnexin (MCC: 0.009±0.003, n =10) or Lamp1 (MCC: 0.067±0.010, n =10). (B) shScr and shStbd1 C2C12 cells transiently transfected with PTG–Myc and double stained for the indicated antibodies. PTG overexpression induces the formation of glycogen structures in the absence of Stbd1 (MCC: shScr, 0.658±0.022, n =10; shStbd1, 0.784±0.029, n =10) which do not coincide with calnexin (MCC: shScr, 0.014±0.002, n =10; shStbd1, 0.107±0.018, n =10). In A and B, representative images are shown. Asterisks indicate non-transfected cells. Inserts show single stainings of the corresponding boxed areas at higher magnification. (C) mRNA expression levels of Ppp1r3c in TM-treated (+) and Stbd1-overexpressing C2C12 myoblasts, as compared to controls [non-treated (−) and GFP-overexpressing cells, respectively]. Data represent mean±s.e.m. of four independent experiments each performed in triplicate. ns, not significant (one-tailed unpaired Student's t -test). Scale bars: 20 μm.

    Journal: Journal of Cell Science

    Article Title: Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts

    doi: 10.1242/jcs.244855

    Figure Lengend Snippet: Stbd1 is not required for PTG-induced glycogen accumulation. (A) C2C12 myoblasts transiently transfected with PTG–Myc and immunostained with the indicated antibodies. PTG overexpression induces the build-up of glycogen clusters strongly colocalizing with PTG [mean±s.e.m. Manders’ colocalization coefficient (MCC): 0.775±0.023, n =10]. PTG-induced glycogen structures do not display any detectable Stbd1 (MCC: 0.024±0.006, n =10) or GS1 (MCC: 0.114±0.012, n =10) immunofluorescence; however, they stain weakly positive for GN (MCC: 0.624±0.013, n =10). No colocalization is observed with calnexin (MCC: 0.009±0.003, n =10) or Lamp1 (MCC: 0.067±0.010, n =10). (B) shScr and shStbd1 C2C12 cells transiently transfected with PTG–Myc and double stained for the indicated antibodies. PTG overexpression induces the formation of glycogen structures in the absence of Stbd1 (MCC: shScr, 0.658±0.022, n =10; shStbd1, 0.784±0.029, n =10) which do not coincide with calnexin (MCC: shScr, 0.014±0.002, n =10; shStbd1, 0.107±0.018, n =10). In A and B, representative images are shown. Asterisks indicate non-transfected cells. Inserts show single stainings of the corresponding boxed areas at higher magnification. (C) mRNA expression levels of Ppp1r3c in TM-treated (+) and Stbd1-overexpressing C2C12 myoblasts, as compared to controls [non-treated (−) and GFP-overexpressing cells, respectively]. Data represent mean±s.e.m. of four independent experiments each performed in triplicate. ns, not significant (one-tailed unpaired Student's t -test). Scale bars: 20 μm.

    Article Snippet: The following primary antibodies were used in this study: anti-Stbd1 (11842-1-AP; Proteintech; 1:500), anti-calnexin (ADI-SPA-860-D; Enzo Life Sciences; 1:250), anti-Lamp1 (1D4B; Developmental Studies Hybridoma Bank; 1:50) and anti-LC3 (NB100-2220; Novus Biologicals; 1:435).

    Techniques: Transfection, Over Expression, Immunofluorescence, Staining, Expressing, One-tailed Test

    ER stress-induced glycogen structures are degraded under conditions of glucose restriction. C2C12 myoblasts were treated with TM for 16 h and cultured for an additional 6 h in medium containing either high (25 mM), low (5 mM) or no glucose, in the absence of TM. Representative images of double immunofluorescence staining for glycogen and Stbd1 are shown. Compared to the glycogen clusters in cells cultured in high-glucose medium, ER stress-induced structures appear smaller under low-glucose conditions and were almost completely devoid of glycogen in the absence of glucose. Despite the near-complete degradation of glycogen under glucose-free conditions, structures induced by ER stress displayed positive immunostaining for Stbd1 [mean±s.e.m. Manders’ colocalization coefficient (MCC): high glucose, 0.764±0.018, n =10; low glucose, 0.772±0.013, n =10; no glucose, 0.202±0.045, n =10]. Overlays are shown as inserts and represent higher magnification of the corresponding boxed areas. Scale bar: 20 μm.

    Journal: Journal of Cell Science

    Article Title: Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts

    doi: 10.1242/jcs.244855

    Figure Lengend Snippet: ER stress-induced glycogen structures are degraded under conditions of glucose restriction. C2C12 myoblasts were treated with TM for 16 h and cultured for an additional 6 h in medium containing either high (25 mM), low (5 mM) or no glucose, in the absence of TM. Representative images of double immunofluorescence staining for glycogen and Stbd1 are shown. Compared to the glycogen clusters in cells cultured in high-glucose medium, ER stress-induced structures appear smaller under low-glucose conditions and were almost completely devoid of glycogen in the absence of glucose. Despite the near-complete degradation of glycogen under glucose-free conditions, structures induced by ER stress displayed positive immunostaining for Stbd1 [mean±s.e.m. Manders’ colocalization coefficient (MCC): high glucose, 0.764±0.018, n =10; low glucose, 0.772±0.013, n =10; no glucose, 0.202±0.045, n =10]. Overlays are shown as inserts and represent higher magnification of the corresponding boxed areas. Scale bar: 20 μm.

    Article Snippet: The following primary antibodies were used in this study: anti-Stbd1 (11842-1-AP; Proteintech; 1:500), anti-calnexin (ADI-SPA-860-D; Enzo Life Sciences; 1:250), anti-Lamp1 (1D4B; Developmental Studies Hybridoma Bank; 1:50) and anti-LC3 (NB100-2220; Novus Biologicals; 1:435).

    Techniques: Cell Culture, Double Immunofluorescence Staining, Immunostaining

    Human neutrophils were isolated from healthy volunteers and then treated with various stimuli for 3 hours in the presence of different gingerol analogues. NETosis was quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). Dose response to LPS-mediated NETosis upon treatment with 6-gingerol ( A ), 8-gingerol ( B ), and 10-gingerol ( C ). NETosis in response to PMA ( D ), RNP ICs ( E ), and APS IgG ( F ) was quantified in the presence of 10 μM gingerol. NETosis was assessed by immunofluorescence microscopy ( G ). Neutrophils were treated with LPS, PMA, RNP ICs, or APS IgG in the presence or absence of 6-gingerol (10 μM). Blue, DNA; green, extracellular neutrophil elastase. Scale bar: 100 microns. For A – F , mean and SEM are presented for n = 3 independent experiments; * P < 0.05, ** P < 0.01, **** P < 0.0001 as compared with the 0 μM gingerol group by 1-way ANOVA corrected with Dunnett’s test.

    Journal: JCI Insight

    Article Title: Antineutrophil properties of natural gingerols in models of lupus

    doi: 10.1172/jci.insight.138385

    Figure Lengend Snippet: Human neutrophils were isolated from healthy volunteers and then treated with various stimuli for 3 hours in the presence of different gingerol analogues. NETosis was quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). Dose response to LPS-mediated NETosis upon treatment with 6-gingerol ( A ), 8-gingerol ( B ), and 10-gingerol ( C ). NETosis in response to PMA ( D ), RNP ICs ( E ), and APS IgG ( F ) was quantified in the presence of 10 μM gingerol. NETosis was assessed by immunofluorescence microscopy ( G ). Neutrophils were treated with LPS, PMA, RNP ICs, or APS IgG in the presence or absence of 6-gingerol (10 μM). Blue, DNA; green, extracellular neutrophil elastase. Scale bar: 100 microns. For A – F , mean and SEM are presented for n = 3 independent experiments; * P < 0.05, ** P < 0.01, **** P < 0.0001 as compared with the 0 μM gingerol group by 1-way ANOVA corrected with Dunnett’s test.

    Article Snippet: In some cases, cultures were also supplemented with 6-gingerol, 8-gingerol, and 10-gingerol (Cayman Chemical).

    Techniques: Isolation, Activity Assay, Immunofluorescence, Microscopy

    Human neutrophils were treated with various stimuli in the presence of different gingerol analogs for 1 hour. Hydrogen peroxide formation was measured by a colorimetric assay. Mean and SEM are presented for n = 3 independent experiments; * P < 0.05, **** P < 0.0001 as compared with the LPS-alone group ( A ), PMA-alone group ( B ), RNP ICs–alone group ( C ), or APS IgG–alone group ( D ) by 1-way ANOVA corrected with Dunnett’s test.

    Journal: JCI Insight

    Article Title: Antineutrophil properties of natural gingerols in models of lupus

    doi: 10.1172/jci.insight.138385

    Figure Lengend Snippet: Human neutrophils were treated with various stimuli in the presence of different gingerol analogs for 1 hour. Hydrogen peroxide formation was measured by a colorimetric assay. Mean and SEM are presented for n = 3 independent experiments; * P < 0.05, **** P < 0.0001 as compared with the LPS-alone group ( A ), PMA-alone group ( B ), RNP ICs–alone group ( C ), or APS IgG–alone group ( D ) by 1-way ANOVA corrected with Dunnett’s test.

    Article Snippet: In some cases, cultures were also supplemented with 6-gingerol, 8-gingerol, and 10-gingerol (Cayman Chemical).

    Techniques: Colorimetric Assay

    Human neutrophils were treated with 6-gingerol. Some samples were additionally treated with forskolin, cAMP, and synthetic PDE4 inhibitors (rolipram and IBMX) as indicated. PDE activity ( A ), cAMP levels ( B ), and PKA activity ( C and D ) were measured with kits as described in Methods. In E , neutrophils were treated with APS IgG in the presence or absence of 6-gingerol and/or PKA inhibitor. NETosis was quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). Mean and SEM are presented for n = 3–4 independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA corrected with Dunnett’s test.

    Journal: JCI Insight

    Article Title: Antineutrophil properties of natural gingerols in models of lupus

    doi: 10.1172/jci.insight.138385

    Figure Lengend Snippet: Human neutrophils were treated with 6-gingerol. Some samples were additionally treated with forskolin, cAMP, and synthetic PDE4 inhibitors (rolipram and IBMX) as indicated. PDE activity ( A ), cAMP levels ( B ), and PKA activity ( C and D ) were measured with kits as described in Methods. In E , neutrophils were treated with APS IgG in the presence or absence of 6-gingerol and/or PKA inhibitor. NETosis was quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). Mean and SEM are presented for n = 3–4 independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA corrected with Dunnett’s test.

    Article Snippet: In some cases, cultures were also supplemented with 6-gingerol, 8-gingerol, and 10-gingerol (Cayman Chemical).

    Techniques: Activity Assay

    BALB/c mice were treated topically with TLR7 agonist (R848) or vehicle DMSO for 6 weeks (3 times per week). Some mice were additionally injected (i.p.) with 20 mg/kg 6-gingerol (3 times per week). Schematic of the TLR7 agonist–induced (R848) lupus model ( A ). NET levels in serum were assessed by measuring cell-free DNA ( B ) and MPO-DNA complexes ( C ). Anti–double-stranded DNA (anti-dsDNA) ( D ), anti–β-2 glycoprotein I (β 2 GPI) IgG ( E ), and total IgG ( F ) levels in serum were assessed by ELISA. Mean is presented as a horizontal line; * P < 0.05, ** P < 0.01, *** P < 0.001 as compared with the R848-alone group by 1-way ANOVA corrected with Dunnett’s test.

    Journal: JCI Insight

    Article Title: Antineutrophil properties of natural gingerols in models of lupus

    doi: 10.1172/jci.insight.138385

    Figure Lengend Snippet: BALB/c mice were treated topically with TLR7 agonist (R848) or vehicle DMSO for 6 weeks (3 times per week). Some mice were additionally injected (i.p.) with 20 mg/kg 6-gingerol (3 times per week). Schematic of the TLR7 agonist–induced (R848) lupus model ( A ). NET levels in serum were assessed by measuring cell-free DNA ( B ) and MPO-DNA complexes ( C ). Anti–double-stranded DNA (anti-dsDNA) ( D ), anti–β-2 glycoprotein I (β 2 GPI) IgG ( E ), and total IgG ( F ) levels in serum were assessed by ELISA. Mean is presented as a horizontal line; * P < 0.05, ** P < 0.01, *** P < 0.001 as compared with the R848-alone group by 1-way ANOVA corrected with Dunnett’s test.

    Article Snippet: In some cases, cultures were also supplemented with 6-gingerol, 8-gingerol, and 10-gingerol (Cayman Chemical).

    Techniques: Injection, Enzyme-linked Immunosorbent Assay

    BALB/c mice were treated topically with TLR7 agonist (R848) or vehicle DMSO for 6 weeks (3 times per week). Starting at week 4 of treatment, some mice were additionally injected (i.p.) with 20 mg/kg 6-gingerol (3 times per week). Schematic of the TLR7 agonist–induced (R848) lupus model by week 4 followed by 6-gingerol treatment ( A ). NET levels in serum were assessed before and after 6-gingerol treatment by measuring MPO-DNA complexes ( B ). Anti–double-stranded DNA (anti-dsDNA) ( C ), anti–β-2 glycoprotein I (β 2 GPI) IgG ( D ), and total IgG ( E ) levels in serum were assessed by ELISA before and after 6-gingerol treatment. Mean is presented as a horizontal line; * P < 0.05, ** P < 0.01, *** P < 0.001 by paired t test.

    Journal: JCI Insight

    Article Title: Antineutrophil properties of natural gingerols in models of lupus

    doi: 10.1172/jci.insight.138385

    Figure Lengend Snippet: BALB/c mice were treated topically with TLR7 agonist (R848) or vehicle DMSO for 6 weeks (3 times per week). Starting at week 4 of treatment, some mice were additionally injected (i.p.) with 20 mg/kg 6-gingerol (3 times per week). Schematic of the TLR7 agonist–induced (R848) lupus model by week 4 followed by 6-gingerol treatment ( A ). NET levels in serum were assessed before and after 6-gingerol treatment by measuring MPO-DNA complexes ( B ). Anti–double-stranded DNA (anti-dsDNA) ( C ), anti–β-2 glycoprotein I (β 2 GPI) IgG ( D ), and total IgG ( E ) levels in serum were assessed by ELISA before and after 6-gingerol treatment. Mean is presented as a horizontal line; * P < 0.05, ** P < 0.01, *** P < 0.001 by paired t test.

    Article Snippet: In some cases, cultures were also supplemented with 6-gingerol, 8-gingerol, and 10-gingerol (Cayman Chemical).

    Techniques: Injection, Enzyme-linked Immunosorbent Assay

    Schematic of the electrolytic model of venous thrombosis ( A ). Direct current results in the release of free radicals within the inferior vena cava, which activate endothelial cells and initiate a thrombogenic environment in the presence of constant blood flow. MPO-DNA complexes were measured in serum of mice treated with control IgG or APS IgG in the presence or absence of 6-gingerol ( B ). Thrombus formation was assessed at 24 hours. Thrombus length ( C ) and thrombus weight ( D ) were measured. Representative thrombi ( E ). ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA corrected with Dunnett’s test.

    Journal: JCI Insight

    Article Title: Antineutrophil properties of natural gingerols in models of lupus

    doi: 10.1172/jci.insight.138385

    Figure Lengend Snippet: Schematic of the electrolytic model of venous thrombosis ( A ). Direct current results in the release of free radicals within the inferior vena cava, which activate endothelial cells and initiate a thrombogenic environment in the presence of constant blood flow. MPO-DNA complexes were measured in serum of mice treated with control IgG or APS IgG in the presence or absence of 6-gingerol ( B ). Thrombus formation was assessed at 24 hours. Thrombus length ( C ) and thrombus weight ( D ) were measured. Representative thrombi ( E ). ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA corrected with Dunnett’s test.

    Article Snippet: In some cases, cultures were also supplemented with 6-gingerol, 8-gingerol, and 10-gingerol (Cayman Chemical).

    Techniques:

    Male C57BL/6 mice were injected (i.p.) with 20 mg/kg 6-gingerol, and peripheral blood was collected after 0.5, 2, 4, and 24 hours. 6-Gingerol concentrations in plasma ( A ) and in neutrophils ( B ) were then quantitated. Values and error bars represent mean ± SEM, respectively.

    Journal: JCI Insight

    Article Title: Antineutrophil properties of natural gingerols in models of lupus

    doi: 10.1172/jci.insight.138385

    Figure Lengend Snippet: Male C57BL/6 mice were injected (i.p.) with 20 mg/kg 6-gingerol, and peripheral blood was collected after 0.5, 2, 4, and 24 hours. 6-Gingerol concentrations in plasma ( A ) and in neutrophils ( B ) were then quantitated. Values and error bars represent mean ± SEM, respectively.

    Article Snippet: In some cases, cultures were also supplemented with 6-gingerol, 8-gingerol, and 10-gingerol (Cayman Chemical).

    Techniques: Injection