Journal: PLOS ONE

Article Title: Astragaloside IV alleviates renal fibrosis by inhibiting renal tubular epithelial cell pyroptosis induced by urotensin II through regulating the cAMP/PKA signaling pathway

doi: 10.1371/journal.pone.0304365

Figure Lengend Snippet: Primer pair sequence of target genes.

Article Snippet: PKA antibodies, Col-I antibodies, FN antibodies, and α-SMA antibodies are bought from Proteintech LTD. IL-1β antibody, NLRP3 antibody, and Caspase-1 antibody are ordered from Beijing Bioss Company.

Techniques: Sequencing

Journal: PLOS ONE

Article Title: Astragaloside IV alleviates renal fibrosis by inhibiting renal tubular epithelial cell pyroptosis induced by urotensin II through regulating the cAMP/PKA signaling pathway

doi: 10.1371/journal.pone.0304365

Figure Lengend Snippet: (A) Effect of AS-IV on α-SMA immunofluorescence expression in renal tubular epithelial cells after UII intervention. (B) Effect of AS-IV on FN immunofluorescence expression in renal tubular epithelial cells after UII intervention. (C) Effect of AS-IV on α-SMA and FN protein expression in renal tubular epithelial cells after UII intervention. (D) Effect of AS-IV on mRNA expression of α-SMA and FN in renal tubular epithelial cells after UII intervention. * means p <0.05 compared with group C, # means p <0.05 compared with group UⅡ. (scale bar: 36.8 μm). The experiment was repeated three times.

Article Snippet: PKA antibodies, Col-I antibodies, FN antibodies, and α-SMA antibodies are bought from Proteintech LTD. IL-1β antibody, NLRP3 antibody, and Caspase-1 antibody are ordered from Beijing Bioss Company.

Techniques: Immunofluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-1 Activates Synthesis of Interleukin-6 by Interfering with a KH-type Splicing Regulatory Protein (KSRP)-dependent Translational Silencing Mechanism *

doi: 10.1074/jbc.M111.264754

Figure Lengend Snippet: mRNAs with increased ribosome association after depletion of KSRP The increase in polysome association (mean of two experiments) of mRNAs from HeLa cells transfected with siRNA against KSRP or against GFP as a control and stimulated with IL-1α for 2 h is shown. Polysome association was calculated as the ratio of signals obtained in the microarray analysis of total RNA of pooled fractions 6–9 (“translated”) over fractions 2–4 (“untranslated”) after gradient centrifugation of cytoplasmic extracts (see B ). GenBank accession numbers are cited in parentheses. cds, coding sequence.

Article Snippet: Reverse transcription and quantitative PCR (RT-qPCR) were carried out as described ( 27 ) using TaqMan kits (Applied Biosystems assay identification numbers Hs00174131_m1 for IL-6 mRNA, Hs00174092_m1 for IL-1α mRNA, Hs99999905_m1 for GAPDH mRNA, and Hs00174103_m1 for IL-8 mRNA and a custom-made assay for rabbit β-globin mRNA).

Techniques: Transfection, Control, Microarray, Gradient Centrifugation, Sequencing, Variant Assay, Virus

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-1 Activates Synthesis of Interleukin-6 by Interfering with a KH-type Splicing Regulatory Protein (KSRP)-dependent Translational Silencing Mechanism *

doi: 10.1074/jbc.M111.264754

Figure Lengend Snippet: The 3′-UTR of IL-6 mRNA suppresses translation. A, cells were transfected with siRNAs specific for GFP or KSRP mRNAs as in Fig. 1 but left without IL-1 treatment. Polysome profiles for endogenous IL-6 mRNA (end. IL-6) or ectopically expressed IL-6 mRNA (ectop. IL-6) were obtained by RT-qPCR. B, distribution profiles of chimeric mRNAs in which the 5′-UTR, coding sequence (CDS), or 3′-UTR of IL-6 was replaced by the corresponding part of β-globin mRNA (see scheme) were obtained by RT-qPCR for IL-6 mRNA or for β-globin mRNA (IL6-B-IL6 and B-B-IL6). The distribution of GAPDH mRNA is shown to allow comparison of the gradient separations. C, cells were transfected with expression vectors for IL-6 with its own (IL6-IL6-IL6) or β-globin 3′-UTR (IL6-IL6-B). The next day medium was exchanged. 2 h later, the culture medium was collected, and IL-6 protein release was quantified by ELISA. The cells were lysed, and IL-6 mRNA was determined by RT-qPCR. Results show the -fold change for the amounts of RNA and protein for IL6-IL6-B as compared with those of IL6-IL6-IL6 (mean ± S.D., n = 5). D, in vitro transcribed IL-6 RNA containing its own (IL6-IL6-IL6) or the β-globin 3′-UTR (IL6-IL6-B) or firefly luciferase RNA were subjected to in vitro translation reactions with reticulocyte lysates and [35S]methionine. The samples were separated by SDS-PAGE, and proteins were visualized by autoradiography. Results from one of two independent assays with similar results are shown.

Article Snippet: Reverse transcription and quantitative PCR (RT-qPCR) were carried out as described ( 27 ) using TaqMan kits (Applied Biosystems assay identification numbers Hs00174131_m1 for IL-6 mRNA, Hs00174092_m1 for IL-1α mRNA, Hs99999905_m1 for GAPDH mRNA, and Hs00174103_m1 for IL-8 mRNA and a custom-made assay for rabbit β-globin mRNA).

Techniques: Transfection, Quantitative RT-PCR, Sequencing, Comparison, Expressing, Enzyme-linked Immunosorbent Assay, In Vitro, Luciferase, SDS Page, Autoradiography

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-1 Activates Synthesis of Interleukin-6 by Interfering with a KH-type Splicing Regulatory Protein (KSRP)-dependent Translational Silencing Mechanism *

doi: 10.1074/jbc.M111.264754

Figure Lengend Snippet: Localization of sequences involved in translational silencing of IL-6 mRNA. A, scheme of IL-6 mRNA and constructs expressed. Numbers indicate nt positions at the ends of deleted or inserted sequences. B, polysome profiles of IL-6 mRNA lacking nt 828–1002 (IL6ΔARE) or containing only nt 816–1024 of its 3′-UTR (IL6 ARE I) were obtained as described for Fig. 2B. For comparison of the gradients, the corresponding profiles of GAPDH mRNA are shown in the lower panel. C, polysome profiles of endogenous IL-6 mRNA (end. IL-6), ectopically expressed IL-6 mRNA containing the complete 3′-UTR (ectop. IL-6), or its derivative containing only nt 857–987 of its 3′-UTR (IL-6ARE II) were compared for cells incubated without (con) or with IL-1α (2 ng/ml) (IL-1) for 1 h. D, comparison of polysome profiles for ectopically expressed IL-6 mRNA without (IL-6) or with mutations that destroy the seed region for miR-126 (IL-6 miR-mu) (upper panel) and for IL-6 miR-mu mRNA in cells incubated without (con) or with IL-1α (IL-1) for 1 h (lower panel).

Article Snippet: Reverse transcription and quantitative PCR (RT-qPCR) were carried out as described ( 27 ) using TaqMan kits (Applied Biosystems assay identification numbers Hs00174131_m1 for IL-6 mRNA, Hs00174092_m1 for IL-1α mRNA, Hs99999905_m1 for GAPDH mRNA, and Hs00174103_m1 for IL-8 mRNA and a custom-made assay for rabbit β-globin mRNA).

Techniques: Construct, Comparison, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-1 Activates Synthesis of Interleukin-6 by Interfering with a KH-type Splicing Regulatory Protein (KSRP)-dependent Translational Silencing Mechanism *

doi: 10.1074/jbc.M111.264754

Figure Lengend Snippet: Direct and ARE-dependent interaction of KSRP with IL-6 mRNA. A, cells expressing Strep-tagged KSRP (stKSRP) or GFP (stGFP) were incubated without (control) or with IL-1α (IL-1) for 30 min and lysed, and tagged proteins were enriched by pulldown with Strep-Tactin-coated beads (see “Experimental Procedures”). Shown are the ratios of the amounts of the indicated mRNAs determined by RT-qPCR in the pulldown and input samples. Note that IL-1α mRNA was not detected in untreated HeLa cells (nd, not detected). B, after stimulation of cells with IL-1α for the indicated times, cytoplasmic and nuclear fractions were prepared and analyzed by Western blotting using antibodies against KSRP, against α-tubulin and ATF1 as controls for fractionation quality, and against NF-κB p65 to control IL-1 responsiveness. C, enrichment of complete IL-6 mRNA or of IL-6 mRNA in which its 3′-UTR was exchanged with that of β-globin (IL6-IL6-B) or that lacks nt 828–1002 (ΔARE) after pulldown with Strep-tagged GFP or Strep-tagged KSRP was determined as in A. D, cytoplasmic (cyt.) lysate or purified Strep-tagged KSRP (200 ng) was incubated with in vitro transcribed radiolabeled RNA comprising the IL-6 3′-UTR with (3′ UTR) or without nt 828 to 1002 (ΔARE). Samples were subjected to non-denaturing gel electrophoresis. The autoradiograph shows protein-RNA complexes and part of the free RNA products.

Article Snippet: Reverse transcription and quantitative PCR (RT-qPCR) were carried out as described ( 27 ) using TaqMan kits (Applied Biosystems assay identification numbers Hs00174131_m1 for IL-6 mRNA, Hs00174092_m1 for IL-1α mRNA, Hs99999905_m1 for GAPDH mRNA, and Hs00174103_m1 for IL-8 mRNA and a custom-made assay for rabbit β-globin mRNA).

Techniques: Expressing, Incubation, Control, Quantitative RT-PCR, Western Blot, Fractionation, Purification, In Vitro, Nucleic Acid Electrophoresis, Autoradiography

Journal: Frontiers in Oncology

Article Title: Interleukin 1 family dysregulation in serum and peritoneal fluid of ovarian cancer patients – potential clinical implications

doi: 10.3389/fonc.2025.1653017

Figure Lengend Snippet: Division of IL-1 family members into subfamilies. Due to the AXD consensus sequence, we can divide the IL-1 family into 3 subfamilies: the IL-1 subfamily (IL-1α, IL-1β, IL-33), the IL-18 subfamily (IL-18, IL-37), the IL-36 subfamily (IL-36α, IL-36β, IL-36γ) and IL-1Ra, which was not included in any of the subfamilies due to the lack of AXD space. On the other hand, the IL-1 family can be divided according to the role it plays in the inflammatory process. Taking this criterion into account, we distinguish seven pro-inflammatory molecules (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ), one anti-inflammatory molecule (IL-37) and three molecules with antagonistic properties (IL-1Ra, IL-36Ra, IL-38) ( – ). Created in https://BioRender.com .

Article Snippet: IL-1α , Human IL-1α ELISA Kit manufactured by Diaclone SAS (Cat. No. 850.005.096) , pg/ml , 10 , 31.25-1000 , CV<4.3% , CV<7.3%.

Techniques: Sequencing

Journal: Frontiers in Oncology

Article Title: Interleukin 1 family dysregulation in serum and peritoneal fluid of ovarian cancer patients – potential clinical implications

doi: 10.3389/fonc.2025.1653017

Figure Lengend Snippet: Summary of signaling pathways of individual IL-1 family members in inflammation. Created in https://BioRender.com . AKT – protein kinase B; AMPK – AMP-activated protein kinase; COX – cyclooxygenase type-2; FAK – focal adhesion kinase; FOXO – forkhead box O; IFN-γ – interferon gamma; IL-6 – interleukin 6; IL-12 – interleukin 12; IRAK – interleukin receptor-associated kinase; MAPK – mitogen-activated protein kinases; MER – myeloid-epithelial-reproductive tyrosine kinase; mTOR – mechanistic target of rapamycin; Myd88 – myeloid differentiation primary response 88; NF-kβ – nuclear factor kβ; p38 – p38 mitogen-activated protein kinases; p53 – tumor protein p53; P62 – nuclear pore glycoprotein p62; Pyk2 – protein tyrosine kinase 2 beta; SHP-2 – K-box region and MADS-box transcription factor family protein; STAT 1, 3 i 6 – signal transducer and activator of transcription 1, 3 i 6; Syk – spleen associated tyrosine kinase; TIR – Toll-interleukin receptor; TNF – tumor necrosis factor; TRAF6 - TNF receptor associated factor 6. IL-1α: Under the influence of proteases, pro-IL-1α is converted into bioactive IL-1α, which, via IL-1R, induces inflammatory responses, affecting neutrophils and monocytes in particular. Subsequently, due to the induction of NF-kβ and MAPK, the inflammatory mediators COX-2, IL-6, TNF are produced, which promote the development of inflammation and further promote the production of IL-1α and IL-1β, enhancing pro-inflammatory processes ( , ). IL-1β: Biologically inactive pro-IL-1β is converted to bioactive IL-1β and subsequently binds to IL-1R. Myd88-, IRAK4-, IRAK6-, TRAF6-mediated signaling is stimulated and the NF-kβ and MAPK pathway is activated, affecting the up-regulation of inflammatory genes . IL-33: IL-33 binds to the specific ST2 receptor and a complex is formed that causes dimerization of TIR. The resulting complex stimulates intracellular signaling through Myd88, IRAK1 and IRAK4 and TRAF6. MAP and NF-kβ are then activated, which stimulate the inflammatory cascade ( , ). IL-18: The inactive pro-IL-18 precursor specifically under the influence of caspase-1 and NLRP3 is converted into biologically active IL-18, which interacts via IL-18R, containing a TIR domain. Subsequently, under the influence of Myd88, IRAK1, IRAK6, TRAF6 are recruited and NF-kβ and the MAPK cascade are activated, which stimulate cells of the immune system. IL-12-mediated synthesis of IFN-γ occurs, leading to the development of the inflammatory process . IL-36α, IL-36β, IL-36γ: Biologically inactive interleukin precursors, under the influence of caspase-1 or specifically by cathepsin G, elastase and proteinase-3, become active molecules that bind to the receptor to form a complex. Myd88 and IRAKs then interact with the complex causing activation of NF-kβ and MAPKs. NF-kβ in the cell nucleus activates pro-inflammatory genes . IL-38 and IL-36Ra: The biological function of these interleukins includes blocking the binding of IL-36 cytokines (IL-36α, IL-36β and IL-36γ) to IL-36R. Due to their antagonistic properties, these interleukins may exhibit anti-inflammatory effects . IL-37: INTRACELLULAR – The inactive pro-IL-37 precursor under the influence of caspase-1 is converted to biologically active IL-37, which forms a complex with Smad3. Down-regulation of individual inflammatory pathways then occurs, preventing the development of inflammation; EXTRACELLULAR – The inactive pro-IL-37 precursor under the influence of caspase-1 is converted into biologically active IL-37, which binds to IL-18Rα and a complex is formed that prevents the activation of inflammatory pathways and thus the development of inflammation ( , ). IL-1Ra: As a secreted form, sIL-1Ra competitively inhibits the binding of IL-1α and IL-1β to the IL-1R1 receptor, thereby inhibiting IL-1-mediated signaling. As an intracellular form, icIL-1Ra1’s signaling mechanism is not entirely clear. It is thought to inhibit NF-kβ activation, by blocking IL-1 binding to IL-1R1 .

Article Snippet: IL-1α , Human IL-1α ELISA Kit manufactured by Diaclone SAS (Cat. No. 850.005.096) , pg/ml , 10 , 31.25-1000 , CV<4.3% , CV<7.3%.

Techniques: Protein-Protein interactions, Produced, Activation Assay, Blocking Assay, Binding Assay

Journal: Frontiers in Oncology

Article Title: Interleukin 1 family dysregulation in serum and peritoneal fluid of ovarian cancer patients – potential clinical implications

doi: 10.3389/fonc.2025.1653017

Figure Lengend Snippet: Comparison of concentrations of individual IL-1 family members: IL-1α (A) , IL-1β (B) , IL-33 (C) , IL-1Ra (D) , IL-18 (E) , IL-37 (F) , IL-36α (G) , IL-36β (H) , IL-36γ (I) , IL-36Ra (J) and IL-38 (K) in serum and peritoneal fluid in patients with benign ovarian tumor (reference group) and patients with ovarian cancer (study group) according to histological differentiation stage (FIGO). In the figure, only data with statistical significance are indicated by arrows. RG - reference group, PF - peritoneal fluid, G1 - highly differentiated, G2 - moderately differentiated, G3 - poorly differentiated, * - p<0.001, ** - p<0.01, *** - p<0.05.

Article Snippet: IL-1α , Human IL-1α ELISA Kit manufactured by Diaclone SAS (Cat. No. 850.005.096) , pg/ml , 10 , 31.25-1000 , CV<4.3% , CV<7.3%.

Techniques: Comparison

Journal: Frontiers in Oncology

Article Title: Interleukin 1 family dysregulation in serum and peritoneal fluid of ovarian cancer patients – potential clinical implications

doi: 10.3389/fonc.2025.1653017

Figure Lengend Snippet: Correlation analysis showing the association between serum and peritoneal fluid IL-1α (A) , IL-1β (B) , IL-33 (C) , IL-1Ra (D) , IL-18 (E) , IL-37 (F) , IL-36α (G) , IL-36β (H) , IL-36γ (I) , IL-36Ra (J) and IL-38 (K) concentrations. Solid line - regression line, dotted line - 95% confidence interval, sign ° - values noted during the analysis, p - level of statistical significance, r - correlation coefficient, PF - peritoneal fluid.

Article Snippet: IL-1α , Human IL-1α ELISA Kit manufactured by Diaclone SAS (Cat. No. 850.005.096) , pg/ml , 10 , 31.25-1000 , CV<4.3% , CV<7.3%.

Techniques:

Journal: Frontiers in Oncology

Article Title: Interleukin 1 family dysregulation in serum and peritoneal fluid of ovarian cancer patients – potential clinical implications

doi: 10.3389/fonc.2025.1653017

Figure Lengend Snippet: (A) IL-1α/IL-1β ratio in the serum from patients in the study and reference groups and the study group depending on the degree of differentiation G1, G2 and G3. (B) IL-1α/IL-1β ratio in the peritoneal fluid from patients in the study group depending on the degree of differentiation G1, G2 and G3. (C) IL-1α/IL-1Ra ratio in the serum from patients in the study and reference groups and the study group depending on the degree of differentiation G1, G2 and G3. (D) IL-1α/IL-1Ra ratio in the peritoneal fluid from patients in the study group depending on the degree of differentiation G1, G2 and G3. (E) IL-1b/IL-Ra ratio in the serum from patients in the study and reference groups and the study group depending on the degree of differentiation G1, G2 and G3. (F) IL-1β/IL-1Ra ratio in the peritoneal fluid from patients in the study group depending on the degree of differentiation G1, G2 and G3.

Article Snippet: IL-1α , Human IL-1α ELISA Kit manufactured by Diaclone SAS (Cat. No. 850.005.096) , pg/ml , 10 , 31.25-1000 , CV<4.3% , CV<7.3%.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Glutaminolysis is required for transforming growth factor-β1–induced myofibroblast differentiation and activation

doi: 10.1074/jbc.RA117.000444

Figure Lengend Snippet: Myofibroblast differentiation and metabolic reprogramming require Gln. A, total cell lysates were isolated from lung fibroblasts treated with or without TGF-β1 (2.5 ng/ml, 0, 24, or 48 h) in the presence or absence of extracellular Gln. Then protein levels of α-SMA, FN, Col1A1, and β-actin were determined by WB; molecular mass markers are indicated on the left side of the panel. B, densitometry analysis of FN, α-SMA, and Col1A1 protein levels determined as described for A. The bar graph summarizes effects of withdrawing Gln from the extracellular culture medium on TGF-β1–induced expression of FN, α-SMA, and Col1A1. The values represent means ± S.E.; the error bars represent S.E. *, p < 0.05 compared with non-differentiated lung fibroblasts grown in the presence of Gln; #, p < 0.05 compared with TGF-β1–treated lung fibroblasts grown in the presence of Gln; ¶, p < 0.05 compared with non-differentiated lung fibroblasts grown in presence of Gln; n.s. stands for non-significant difference between means (n = 3; 3 biological replicates total, 1 technical replicate per group per experiment). C, total cell lysates were isolated from lung fibroblasts treated with or without TGF-β1 (2.5 ng/ml, 24 h) and switched or not to Gln-free medium for 24 h post–TGF-β1 treatment. Then protein levels of FN, HIF-1α, α-SMA, and GAPDH were determined by WB; molecular mass markers are indicated on the left side of the panel. D, densitometry analysis of FN, HIF-1α, and α-SMA protein levels determined as described for C. The bar graph summarizes effects of withdrawing Gln from the extracellular culture medium post–TGF-β1 treatment on the expression of FN, HIF-1α, and α-SMA. The values represent means ± S.E.; the error bars represent S.E. *, p < 0.05 compared with non-differentiated lung fibroblasts grown in presence of Gln; #, p < 0.05 compared with TGF-β1–treated lung fibroblasts grown in presence of Gln; n.s. stands for non-significant difference between means (n = 3; 3 biological replicates total, 1 technical replicate per group per experiment). E, bioenergetic map of lung fibroblasts representing the shift in OCR and ECAR following the switch of cells treated with or without TGF-β1 to Gln-free medium.

Article Snippet: SMAD3 (clone C67H9, rabbit mAb against amino residues within the amino terminus of SMAD3; catalog no. 9523, lot no. 7), HIF-1α (clone D2U3T, rabbit mAb against residues surrounding Lys-460 of human HIF-1α; catalog no. 14179, lot no. 1), and GAPDH (clone D16H11, rabbit mAb against residues near the carboxyl terminus of human GAPDH; catalog no. 5174S, lot no. 4) antibodies were purchased from Cell Signaling Technology.

Techniques: Isolation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Glutaminolysis is required for transforming growth factor-β1–induced myofibroblast differentiation and activation

doi: 10.1074/jbc.RA117.000444

Figure Lengend Snippet: GLS1 silencing prevent TGF-β1–induced expression of profibrotic markers. A, total cell lysates were isolated from lung fibroblasts transfected with NT or GLS1 siRNA (100 nm) and treated with or without TGF-β1 (2.5 ng/ml, 48 h). Then protein levels of α-SMA, FN, Col1A1, HIF-1α, GLS1, and β-actin were determined by WB; molecular mass markers are indicated on the left side of the panel. B–F, densitometry analyses of total protein levels of α-SMA, FN, Col1A1, HIF-1α, and GLS1 determined as described for A. The values represent means ± S.E.; the error bars represent S.E. *, p < 0.01 compared with NT transfected non-differentiated lung fibroblasts; #, p < 0.05 compared with NT transfected TGF-β1–treated lung fibroblasts; ¶, p < 0.005 compared with NT transfected non-differentiated lung fibroblasts (n = 5; 5 biological replicates total, 1 technical replicate per group per experiment).

Article Snippet: SMAD3 (clone C67H9, rabbit mAb against amino residues within the amino terminus of SMAD3; catalog no. 9523, lot no. 7), HIF-1α (clone D2U3T, rabbit mAb against residues surrounding Lys-460 of human HIF-1α; catalog no. 14179, lot no. 1), and GAPDH (clone D16H11, rabbit mAb against residues near the carboxyl terminus of human GAPDH; catalog no. 5174S, lot no. 4) antibodies were purchased from Cell Signaling Technology.

Techniques: Expressing, Isolation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Glutaminolysis is required for transforming growth factor-β1–induced myofibroblast differentiation and activation

doi: 10.1074/jbc.RA117.000444

Figure Lengend Snippet: Exogenous glutamate or α-KG restores the expression of profibrotic markers in GLS1-deficient myofibroblasts. A, total cell lysates were isolated from lung fibroblasts transfected with NT (lanes N) or GLS1 (lanes G) siRNA (100 nm), treated with or without l-glutamic acid (2 mm), and treated with or without TGF-β1 (2.5 ng/ml, 48 h). l-Glutamic acid was added in the reduced serum DMEM (0.5% fetal calf serum supplemented with 2 mm l-glutamine) 1 h prior to the addition of TGF-β1. Then protein levels of α-SMA, HIF-1α, GLS1, and α-tubulin were determined by WB; molecular mass markers are indicated on the left side of the panel (representative WB of n = 3). B, total cell lysates were isolated from lung fibroblasts transfected with NT or GLS1 siRNA (100 nm), treated with or without diethyl-2-oxopentanedioate (esterified form of α-KG, 2 mm), and treated with or without TGF-β1 (2.5 ng/ml, 48 h). Diethyl-2-oxopentanedioate was added in the reduced serum DMEM (0.5% fetal calf serum supplemented with 2 mm l-glutamine) 24 h prior to the addition of TGF-β1. Then protein levels of α-SMA, HIF-1α, GLS1, and α-tubulin were determined by WB; molecular mass markers are indicated on the left side of the panel (representative WB of n = 3).

Article Snippet: SMAD3 (clone C67H9, rabbit mAb against amino residues within the amino terminus of SMAD3; catalog no. 9523, lot no. 7), HIF-1α (clone D2U3T, rabbit mAb against residues surrounding Lys-460 of human HIF-1α; catalog no. 14179, lot no. 1), and GAPDH (clone D16H11, rabbit mAb against residues near the carboxyl terminus of human GAPDH; catalog no. 5174S, lot no. 4) antibodies were purchased from Cell Signaling Technology.

Techniques: Expressing, Isolation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Glutaminolysis is required for transforming growth factor-β1–induced myofibroblast differentiation and activation

doi: 10.1074/jbc.RA117.000444

Figure Lengend Snippet: siRNA sequences

Article Snippet: SMAD3 (clone C67H9, rabbit mAb against amino residues within the amino terminus of SMAD3; catalog no. 9523, lot no. 7), HIF-1α (clone D2U3T, rabbit mAb against residues surrounding Lys-460 of human HIF-1α; catalog no. 14179, lot no. 1), and GAPDH (clone D16H11, rabbit mAb against residues near the carboxyl terminus of human GAPDH; catalog no. 5174S, lot no. 4) antibodies were purchased from Cell Signaling Technology.

Techniques: Sequencing

Journal: Communications Biology

Article Title: βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity

doi: 10.1038/s42003-021-01763-5

Figure Lengend Snippet: Representative western blot and densitometry graph from co-immunoprecipitation studies in mouse WT ( a , b ) or Cryba1 KO ( c , d ) astrocytes transfected with blank-mCherry, βA3-crystallin-mCherry (βA3-mCherry), βA1-crystallin-mCherry (βA1-mCherry), and βA3/A1-crystallin-mCherry (βA3/A1-mCherry) show interaction of PTP1B with both βA3- and βA1-crystallin; n = 4. e , f Co-immunoprecipitation assay showing βA3/A1-crystallin levels in the Co-IP eluent by western blot analysis and densitometry, indicating binding to PTP1B, upon pull down with mNeonGreen antibody-bound magnetic beads from lysates of astrocytes overexpressing PTP1B (Ad-CMV-mNeonGreen-m Ptpn1 ) and Cryba1 (Ad-CMV-RFP-m Cryba1 ). Pull down with mouse IgG showed no binding for βA3/A1-crystallin; n = 4. g Ribbon diagram obtained by molecular modeling showing superimposed βA1-crystallin (orange), βA3-crystallin (blue), and PTP1B (gray). Neither isoform is able to bind to the pocket of PTP1B active site (Cys215, Red). The βA1, but not the βA3 isoform (due to a steric bump of terminal extension) interacts with an allosteric binding site (green) on the surface of PTP1B.

Article Snippet: Primary antibodies: Phospho-Stat3 (Tyr705) (Thermo Fisher, USA; Cat# 44380G), p-NFκB p65 (S536) (Thermo Fisher, USA; Cat# MA515160), STAT3 (Thermo Fisher, USA; Cat# 10253-2-AP), beta Crystallin A3 (Abcam, USA; Cat# ab151722), IL-6 (Biorbyt, USA; Cat# orb6210), IL-1α (Biorbyt, USA; Cat# orb184287) and mNeonGreen (Chromotek, USA; Cat# 3216-100), Secondary antibodies: HRP anti-Rabbit IgG (KPL, USA; Cat# 074-1506), HRP anti-tagged anti-Mouse IgG (KPL, USA; Cat# 5220-0341), HRP anti-tagged Goat IgG (KPL, USA; Cat# 14-13-06).

Techniques: Western Blot, Immunoprecipitation, Transfection, Co-Immunoprecipitation Assay, Binding Assay, Magnetic Beads

Journal: Communications Biology

Article Title: βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity

doi: 10.1038/s42003-021-01763-5

Figure Lengend Snippet: a Lineweaver-Burk plot showing increasing doses (0, 0.5, 1, and 2 nM) of βA3/A1-crystallin decreases both V max and K M of PTP1B activity for different concentrations of p-Nitrophenyl Phosphate (pNPP). n = 3. * P < 0.05. ** P < 0.05. b The N-terminal sequence of Cryba1 ; βA1-crystallin KD mice were generated by knocking in 5 base pairs (CCACC, red) before the first start codon to strengthen the Kozak consensus sequence. For generating βA3-crystallin KO, the first start codon was removed by a single nucleotide mutation in the mouse Cryba1 gene (A > G, red). Another silent mutation (C > G; red) was also introduced to prevent the binding and re-cutting of the sequence by gRNA after homology-directed repair. c Representative western blot and d graph showing densitometry analysis for the expression level of βA3/A1-crystallin in astrocyte lysates from WT (black bar), βA3 KO (blue bar), and βA1 KD (green bar) mice, respectively, showing complete loss of βA3-crystallin in the βA3 KO cells and a notable decrease in βA1-crystallin expression in the βA1 KD cells, relative to WT astrocytes. In βA3 KO astrocytes, there is an increase in expression of βA1-crystallin; n = 4. * P < 0.05, ** P < 0.01. e Increased levels of lactate in mouse WT, βA3 KO and βA1 KD astrocytes treated with HG (25 or 30 mM for 6 h) relative to untreated cells. Lactate levels in βA1 KD astrocytes were higher in all experimental conditions, compared to WT and βA3 KO cells; n = 3. * P < 0.05, ** P < 0.01. f Elevated glycolytic flux is evident from increased glycolytic capacity in WT, βA3 KO and βA1 KD astrocytes treated with high glucose (HG; 30 mM for 6 h), relative to untreated cells (cultured in 5 mM d -glucose containing medium). Glycolytic capacity in βA1 KD astrocytes was drastically higher compared to WT and βA3 KO cells; n = 4. * P < 0.05, ** P < 0.01. g Cultured βA1 astrocytes either untreated or exposed to mannitol (30 mM for 6 h) have elevated PTP1B activity compared to WT and βA3 KO cells, which increases further with HG (30 mM for 6 h). The elevation in PTP1B activity was rescued by βA1-crystallin overexpression in untreated or HG-exposed βA1 KD cells; n = 5. * P < 0.05, ** P < 0.01.

Article Snippet: Primary antibodies: Phospho-Stat3 (Tyr705) (Thermo Fisher, USA; Cat# 44380G), p-NFκB p65 (S536) (Thermo Fisher, USA; Cat# MA515160), STAT3 (Thermo Fisher, USA; Cat# 10253-2-AP), beta Crystallin A3 (Abcam, USA; Cat# ab151722), IL-6 (Biorbyt, USA; Cat# orb6210), IL-1α (Biorbyt, USA; Cat# orb184287) and mNeonGreen (Chromotek, USA; Cat# 3216-100), Secondary antibodies: HRP anti-Rabbit IgG (KPL, USA; Cat# 074-1506), HRP anti-tagged anti-Mouse IgG (KPL, USA; Cat# 5220-0341), HRP anti-tagged Goat IgG (KPL, USA; Cat# 14-13-06).

Techniques: Activity Assay, Sequencing, Generated, Mutagenesis, Binding Assay, Western Blot, Expressing, Cell Culture, Over Expression

Journal: Communications Biology

Article Title: βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity

doi: 10.1038/s42003-021-01763-5

Figure Lengend Snippet: a , b Plots from seahorse analysis using the mitostress assay showing time-dependent changes in metabolic flux upon treatment with mitochondrial respiration blockers oligomycin, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), Rotenone/Antimycin A at particular time points (indicated by arrows). Y -axis denotes oxygen consumption rate (OCR; pmole/min/μg) and X -axis represents time (minutes) for a untreated (cultured in 5 mM d -Glucose) WT, βA3 KO, and βA1 KD mouse astrocytes or b astrocytes exposed to either mannitol (30 mM for 6 h) or high glucose (HG; 30 mM for 6 h) respectively, or βA1 astrocytes transfected with mCherry-βA1 overexpression construct for 48 h or treated with 10 μM of PTP1B inhibitor (MSI-1436) for 1 h prior to HG (30 mM) exposure for 6 h; n = 4. Reduced mitochondrial function is shown by decreased c ATP-linked respiration and d maximal respiration, in untreated or mannitol (30 mM for 6 h) and HG (30 mM for 6 h)-exposed βA1 KD astrocytes, compared to WT cells. WT and βA3 KO astrocytes treated with HG showed an increase in both c ATP-linked respiration and d maximal respiration, compared to untreated cells. In βA1 KD astrocytes transfected with βA1-mCherry overexpression construct for 48 h or treated with 10 μM of PTP1B inhibitor (MSI-1436) for 1 h prior to HG (30 mM) exposure for 6 h, the levels of c ATP-linked respiration and d maximal respiration were partially rescued; n = 4. * P < 0.05, ** P < 0.01. e , f Flow cytometry histograms and graph for MitoSox fluorescence (Alexa fluor (AF)-555), g Nox1 gene expression, h Nox2 gene expression and i superoxide release in βA1 KD astrocytes either untreated or exposed to mannitol (30 mM for 6 h) showed increased levels of e , f mROS, g Nox1 along with h Nox2 gene expression and i superoxide release, that increased further with HG (30 mM for 6 h) exposure, relative to WT cells ( e – h ). βA1-crystallin overexpression (using βA1-mCherry construct) or PTP1B inhibition in βA1 KD astrocytes followed by treatment with HG, reduced the elevated levels of e , f mROS, g Nox1 , and h Nox2 gene expression and i superoxide release in βA1 KD cells. n = 4. * P < 0.05, ** P < 0.01.

Article Snippet: Primary antibodies: Phospho-Stat3 (Tyr705) (Thermo Fisher, USA; Cat# 44380G), p-NFκB p65 (S536) (Thermo Fisher, USA; Cat# MA515160), STAT3 (Thermo Fisher, USA; Cat# 10253-2-AP), beta Crystallin A3 (Abcam, USA; Cat# ab151722), IL-6 (Biorbyt, USA; Cat# orb6210), IL-1α (Biorbyt, USA; Cat# orb184287) and mNeonGreen (Chromotek, USA; Cat# 3216-100), Secondary antibodies: HRP anti-Rabbit IgG (KPL, USA; Cat# 074-1506), HRP anti-tagged anti-Mouse IgG (KPL, USA; Cat# 5220-0341), HRP anti-tagged Goat IgG (KPL, USA; Cat# 14-13-06).

Techniques: Cell Culture, Transfection, Over Expression, Construct, Flow Cytometry, Fluorescence, Gene Expression, Inhibition

Journal: Communications Biology

Article Title: βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity

doi: 10.1038/s42003-021-01763-5

Figure Lengend Snippet: a Representative western blot and b , c densitometry graphs showing decreased phosphorylation of STAT3 at tyrosine 705 (p-STAT3 Y705 ) in βA1 KD astrocytes b untreated or c treated with 30 mM mannitol or high glucose (HG; 30 mM) for 6 h, relative to WT cells ( c ). βA3 KO cells did not show such changes ( a – c ); n = 4. ** P < 0.01. d , e Overexpression of βA1-crystallin (using βA1-mCherry construct) in βA1 KD astrocytes or treatment with 10 μM PTP1B inhibitor (MSI-1436) for 1 h, prior to HG (30 mM) exposure for 6 h rescued the levels of p-STAT3 Y705 , as compared to βA1 KD cells transfected with blank-mCherry construct. βA1-crystallin overexpression was confirmed by mCherry western blot. f Western blot and g densitometry graph showing decrease in p-STAT3 Y705 expression in WT cells infected with Adenovirus-PTP1B overexpression construct or Cryba1 shRNA for 48 h, followed by HG treatment. h Cryba1 knockdown was confirmed by qPCR, which showed about 70% downregulation compared to control. n = 4. * P < 0.05, ** P < 0.01. i , j Representative images from live-cell confocal microscopy of human iPSC-derived astrocytes after overexpression of i βA1-mCherry or j βA3-mCherry constructs and k quantitative assessment of nuclear translocation, showing nuclear localization of βA1-crystallin in these cells ( i , k ), whereas βA3-crystallin construct transfected cells showed less nuclear localization ( j , k ). Scale bar, 10 μm. ** P < 0.01 ( n = 16).

Article Snippet: Primary antibodies: Phospho-Stat3 (Tyr705) (Thermo Fisher, USA; Cat# 44380G), p-NFκB p65 (S536) (Thermo Fisher, USA; Cat# MA515160), STAT3 (Thermo Fisher, USA; Cat# 10253-2-AP), beta Crystallin A3 (Abcam, USA; Cat# ab151722), IL-6 (Biorbyt, USA; Cat# orb6210), IL-1α (Biorbyt, USA; Cat# orb184287) and mNeonGreen (Chromotek, USA; Cat# 3216-100), Secondary antibodies: HRP anti-Rabbit IgG (KPL, USA; Cat# 074-1506), HRP anti-tagged anti-Mouse IgG (KPL, USA; Cat# 5220-0341), HRP anti-tagged Goat IgG (KPL, USA; Cat# 14-13-06).

Techniques: Western Blot, Phospho-proteomics, Over Expression, Construct, Transfection, Expressing, Infection, shRNA, Knockdown, Control, Confocal Microscopy, Derivative Assay, Translocation Assay

Journal: Communications Biology

Article Title: βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity

doi: 10.1038/s42003-021-01763-5

Figure Lengend Snippet: a Representative western blot and b densitometry graph showing increased phosphorylation of NFκB at serine 536 (p-NFκB S536 ) in untreated (cultured in 5 mM d -Glucose containing medium) βA1 KD astrocytes, relative to WT cells. βA3 KO cells did not show such change in p-NFκB S536 levels. n = 4. * P < 0.05. c , d βA1 KD astrocytes transfected with βA1-mCherry construct for 48 h or treated with 10 μM of PTP1B inhibitor (MSI-1436) for 1 h, prior to HG (30 mM) exposure for 6 h rescued the levels of p-NFκB S536 , compared to βA1 KD cells transfected with blank mCherry construct and then exposed to HG. βA1-crystallin overexpression was confirmed by mCherry western blot. n = 4. * P < 0.05, ** P < 0.01. e IL-6 and f IL-1α levels showed marked increase in HG-exposed WT, βA3 KO, and βA1 KD astrocytes, compared to untreated cells. βA1 KD astrocytes showed increased levels of both cytokines relative to WT or βA3 KO cells. ( e , f ) Overexpression of βA1-crystallin (using βA1-mCherry construct) in βA1 KD astrocytes or treatment with 10 μM of PTP1B inhibitor (MSI-1436) for 1 h, prior to HG (30 mM) exposure for 6 h, rescued the levels of IL-6 and IL-1α. n = 4. * P < 0.05, ** P < 0.01.

Article Snippet: Primary antibodies: Phospho-Stat3 (Tyr705) (Thermo Fisher, USA; Cat# 44380G), p-NFκB p65 (S536) (Thermo Fisher, USA; Cat# MA515160), STAT3 (Thermo Fisher, USA; Cat# 10253-2-AP), beta Crystallin A3 (Abcam, USA; Cat# ab151722), IL-6 (Biorbyt, USA; Cat# orb6210), IL-1α (Biorbyt, USA; Cat# orb184287) and mNeonGreen (Chromotek, USA; Cat# 3216-100), Secondary antibodies: HRP anti-Rabbit IgG (KPL, USA; Cat# 074-1506), HRP anti-tagged anti-Mouse IgG (KPL, USA; Cat# 5220-0341), HRP anti-tagged Goat IgG (KPL, USA; Cat# 14-13-06).

Techniques: Western Blot, Phospho-proteomics, Cell Culture, Transfection, Construct, Over Expression

Journal: Communications Biology

Article Title: βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity

doi: 10.1038/s42003-021-01763-5

Figure Lengend Snippet: ELISA analysis from total mouse retinal lysates showed increased a IL-6 and b IL-1α in diabetic (STZ-treated) WT, βA3 KO, and especially in βA1 KD mice relative to non-diabetic littermates. In diabetic βA1 KD mice, intraperitoneal treatment (thrice weekly) with PTP1B inhibitor (MSI-1436), at a dose of 0.125 mg/kg body weight started 3 weeks after diabetes onset and continued for 5 weeks or a single intravitreal injection of AAV2- Cryba1 construct (1.64 × 10 12 vg/ml), rescued the IL-6 and IL-1α levels. n = 4. ** P < 0.01. c – l Representative images of the retinal capillary network and quantitative graph to show the number of acellular capillaries ( m ), in diabetic and non-diabetic WT, βA3 KO and βA1 KD mice, showing increase in acellular capillaries (arrows) in non-diabetic ( e , inset zoomed in f ) or diabetic βA1 KD mice ( i , inset zoomed in j ), compared to WT mice ( c , g , and m ). βA3 KO mice ( d , h ) did not show any noticeable change in retinal vasculature relative to WT mice. MSI-1436 or AAV2- Cryba1 treatment reduced degenerative changes in the retinal vasculature (arrows in k and l , graph m ) in βA1 KD animals. n = 4. * P < 0.05, ** P < 0.01. Scale bar, 50 μm ( c – e , g – i , k – l ). Scale bar, 100 μm (insets, f and j ).

Article Snippet: Primary antibodies: Phospho-Stat3 (Tyr705) (Thermo Fisher, USA; Cat# 44380G), p-NFκB p65 (S536) (Thermo Fisher, USA; Cat# MA515160), STAT3 (Thermo Fisher, USA; Cat# 10253-2-AP), beta Crystallin A3 (Abcam, USA; Cat# ab151722), IL-6 (Biorbyt, USA; Cat# orb6210), IL-1α (Biorbyt, USA; Cat# orb184287) and mNeonGreen (Chromotek, USA; Cat# 3216-100), Secondary antibodies: HRP anti-Rabbit IgG (KPL, USA; Cat# 074-1506), HRP anti-tagged anti-Mouse IgG (KPL, USA; Cat# 5220-0341), HRP anti-tagged Goat IgG (KPL, USA; Cat# 14-13-06).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Construct

Journal: Cancer Research

Article Title: Phagocytosis of Glioma Cells Enhances the Immunosuppressive Phenotype of Bone Marrow–Derived Macrophages

doi: 10.1158/0008-5472.CAN-22-1570

Figure Lengend Snippet: Immunosuppressive phenotype of double-positive TAMs is caused by phagocytosis of glioma cells. A, Schematic workflow for the RNA-seq of BMDMs. Top, BMDMs cocultured with GSCs for 24 hours. Bottom, BMDMs were pretreated with cytoD for 1 hour to inhibit their phagocytic abilities, then cocultured with GSCs for 24 hours. B, GSEA plots of BMDMs treated with cytoD before coculture compared with BMDMs cocultured with GSCs directly. GSEA was performed by the R package clusterProfiler. C, Bar plots showing the expression levels of proinflammatory genes. Bars are colored according to treatments. Data, mean ± SEM. P values were calculated by two-sided Student t test. D, Heat map depicting the differentially expressed genes following different treatments. E, Comparison of IL1α, IL1β, TNFα, and IL10 levels in M1 BMDMs under different coculture conditions. M1, M1 macrophages cultured alone; M1 + NSC, M1 macrophages cocultured with NSCs; M1 + GSC, M1 macrophages cocultured with GSCs; M1 + GSC#, M1 macrophages cocultured with GSCs by Transwell with 0.4 μm pore; cytoD-M1 + GSC, M1 macrophages pretreated with cytoD, then cocultured with GSCs. Data, mean + SEM. P values were calculated by two-sided Student t test. F, Comparison of mean fluorescence intensity (MFI) of CD163, CD206, CD276, PD-L1, and PD-L2 in M1 BMDMs under different coculture conditions. M1, M1 macrophages cultured alone; M1 + NSC, M1 macrophages cocultured with NSCs; M1 + GSC, M1 macrophages cocultured with GSCs; M1 + GSC#, M1 macrophages cocultured with GSCs by Transwell with 0.4 μm pore; cytoD-M1 + GSC, M1 macrophages pretreated with cytoD, then cocultured with GSCs. Data, mean + SEM. P values were calculated by two-sided Student t test. G, Percentage of proliferative T cells under different treatments. #, cells cultured by Transwell with 0.4 μm pore. Data, mean ± SEM. P values are calculated by a two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The protein levels of IL1α, IL1β, TNFα, and IL10 in the macrophage culture supernatant were measured with Human IL1α ELISA Kit (Cusabio, #CSB-E04620h), Human IL1β ELISA Kit (Cusabio, #CSB-E08053h), Human TNFα ELISA Kit (Cusabio, #CSB-E04740h), and Human IL10 ELISA Kit (Cusabio, #CSB-E04593h), respectively.

Techniques: RNA Sequencing, Expressing, Comparison, Cell Culture, Fluorescence

Journal: Cancer Research

Article Title: Phagocytosis of Glioma Cells Enhances the Immunosuppressive Phenotype of Bone Marrow–Derived Macrophages

doi: 10.1158/0008-5472.CAN-22-1570

Figure Lengend Snippet: Immunosuppressive activity of double-positive TAMs. A, Pathways that downregulated in GFP + F4/80 + BMDMs, as compared with GFP – F4/80 + BMDMs. B, GSEA plot of GFP + F4/80 + BMDMs compared with GFP – F4/80 + BMDMs. GSEA was performed by the R package clusterProfiler. C, Comparison of IL1α, IL1β, TNFα, and IL10 levels in M1 BMDMs under different coculture conditions. M1, M1 macrophages cultured alone; CD45 + GFP + , M1 macrophages with GFP fluorescence; CD45 + GFP – , M1 macrophages without GFP fluorescence. Data, mean + SEM. P values were calculated by a two-sided Student t test. D, Comparison of mean fluorescence intensity (MFI) of CD163, CD206, CD276, PD-L1, and PD-L2 in M1 BMDMs under different coculture conditions. Data, mean + SEM. P values are calculated by two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Reg., regulation.

Article Snippet: The protein levels of IL1α, IL1β, TNFα, and IL10 in the macrophage culture supernatant were measured with Human IL1α ELISA Kit (Cusabio, #CSB-E04620h), Human IL1β ELISA Kit (Cusabio, #CSB-E08053h), Human TNFα ELISA Kit (Cusabio, #CSB-E04740h), and Human IL10 ELISA Kit (Cusabio, #CSB-E04593h), respectively.

Techniques: Activity Assay, Comparison, Cell Culture, Fluorescence