Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on tumor necrosis factor (TNF)-α protein expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. Protein expression levels of TNF-α were determined using immunohistochemistry (magnification, ×400). (A) No TNF-α expression was detected in the Schwann cells derived from the SN of a sham-operated rat. (B) Moderate protein expression levels of TNF-α were detected in the Schwann cells derived from the SN fiber of a rat in the 12 h post-reperfusion I/R subgroup. (C) Higher protein expression levels of TNF-α were detected in the Schwann cells derived from the SN of a rat in the 24 h post-reperfusion I/R subgroup. (D) Numerous inflammatory cells had infiltrated the area surrounding the Schwann cells and moderate protein expression levels of TNF-α were detected in the SN of a rat in the 72 h post-reperfusion I/R subgroup. (E) Widespread demyelination and mild-to-moderate TNF-α protein expression levels in Schwann cells were detected in the SN derived from a rat in the 7 days post-reperfusion I/R subgroup. (F) A SN from a rat in the I/R + MK-801 group at 12 h post-reperfusion exhibited mild-to-moderate TNF-α protein expression levels in Schwann cells. (G) A SN from a rat in the I/R + MK-801 group at 24 h post-reperfusion exhibited markedly fewer infiltrating cells, as compared with the SN derived from I/R rats at the same time point post-reperfusion. Moderate protein expression levels of TNF-α expression were observed. (H) A SN derived from a rat in the I/R + MK-801 group at 7 days post-reperfusion. As compared with the SNs derived from the I/R rats, the extent of demyelination was markedly reduced and Schwann cells exhibited only low protein expression levels of TNF-α.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Protein expression levels of tumor necrosis factor-α in the various treatment subgroups were quantified using the integrated optical density method, and are presented as the mean ± standard deviation (n=6). Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. I/R, ischemia reperfusion.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on TNF-α and TACE mRNA expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. TNF-α and TACE mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction and are expressed relative to β-actin. Agarose gel images showing TNF-α and TACE mRNA expression levels in the SN homogenates at (A) 0, (B) 6, (C) 12, (D) 24 and (E) 72 h and (F) 7 days post-reperfusion. β-actin=280 bp; TNF-α=402 bp; TACE=624 bp. Relative (G) TNF-α and (H) TACE mRNA expression levels are presented as the mean ± standard deviation (n=6). *P<0.05, **P<0.01 vs. the sham-operated group; Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. TNF-α, tumor necrosis factor-α; TACE, TNF-α-converting enzyme.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Standard Deviation

Journal: Cancer gene therapy

Article Title: A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

doi: 10.1038/sj.cgt.7700068

Figure Lengend Snippet: Figure 1. Ad vectors containing TNFa expression cassettes. Both Ad-75 (Ad- mTNF-75) and Ad-hTNF (Ad-MCMV- TNF)25 vectors express the mature mTNFa and hTNFa cDNAs, respec- tively, fused with the IFN-g signal pep- tide. Ad-mTNF (Ad-mTNF-wt)24 con- tains the entire mTNFa cDNA. All TNFa-expressing vectors use the MCMV promoter and an SV40 polyad- enylation signal. All vectors are E1/E3- deleted Ad-5-based vectors.

Article Snippet: Western blotting was done by blocking the membrane with 5% powdered milk in PBS and subsequently incubating with primary polyclonal goat anti-mouse TNFa Ab (R&D Systems) for 4 hours at room temperature or overnight at 4°C.

Techniques: Expressing

Journal: Cancer gene therapy

Article Title: A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

doi: 10.1038/sj.cgt.7700068

Figure Lengend Snippet: Figure 2. TNF expression in Ad-vector-transduced cell supernatants: detection of mutant (D142N-A144R) mTNFa expression by Western blot with a polyclonal goat anti-mouse TNFa Ab. Expression was detected from the supernatants of the Ad-mTNF- or Ad-75-transduced MRC5, NDL, and MT1A2 (1A2) cell lines as described in Materials and Methods. Samples (12.5 mL of supernatant per well) were mixed 50:50 with loading buffer. Ad-BHGDE1,3-transduced MT1A2 cells were used as a negative control.

Article Snippet: Western blotting was done by blocking the membrane with 5% powdered milk in PBS and subsequently incubating with primary polyclonal goat anti-mouse TNFa Ab (R&D Systems) for 4 hours at room temperature or overnight at 4°C.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Negative Control

Journal: Cancer gene therapy

Article Title: A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

doi: 10.1038/sj.cgt.7700068

Figure Lengend Snippet: Figure 3. In vitro bioactivity of mutant mTNFa. A: Cytotoxicity (MTT) assay on TNFa-sensitive L929 cells with 10 mL of supernatants from Ad-75- (75), Ad-mTNF- (mTNF), Ad-hTNF- (hTNF), and Ad-BHGDE1,3- (E1,3) transduced MRC5, NDL, and MT1A2 cells (MOI 5 50 PFU/cell). The cytotoxicity of standard dilutions of recombinant mTNFa is indicated by the line graph on the right. B: Proliferation assay on the TNFa-responsive CT6 cell line (detected by MTT), treated with 10 mL of supernatants from Ad vector-transduced MT1A2 cells (MOI 5 50 PFU/cell). Solid bars represent wells treated with soluble murine p55 TNFR (33 mg/mL). The proliferation of CT6 cells in response to recombinant mTNFa is indicated by the cross-hatched bars on the right.

Article Snippet: Western blotting was done by blocking the membrane with 5% powdered milk in PBS and subsequently incubating with primary polyclonal goat anti-mouse TNFa Ab (R&D Systems) for 4 hours at room temperature or overnight at 4°C.

Techniques: In Vitro, Mutagenesis, MTT Assay, Recombinant, Proliferation Assay, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

doi: 10.1038/sj.cgt.7700068

Figure Lengend Snippet: Figure 4. In vitro binding of mutant and wt TNFa to TNFR: coimmunoprecipitation/Western blot of wt hTNFa and mTNFa and mutant (D142N-A144R) mTNFa in 500 mL of culture supernatant from NDL cells transduced with the respective Ad vectors (MOI 5 50 PFU/cell) using soluble murine p55 and p75 TNFRs (2 mg per precipitation), as described in Materials and Methods. Receptors were immunoprecipitated by specific Abs that did not interfere with ligand binding (2 mg per precipitation). Coimmunoprecipitates were analyzed by Western blot, first for mTNFa, and second for hTNFa (see Materials and Methods). sTNFR refers to soluble TNFR.

Article Snippet: Western blotting was done by blocking the membrane with 5% powdered milk in PBS and subsequently incubating with primary polyclonal goat anti-mouse TNFa Ab (R&D Systems) for 4 hours at room temperature or overnight at 4°C.

Techniques: In Vitro, Binding Assay, Mutagenesis, Western Blot, Transduction, Immunoprecipitation, Ligand Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

doi: 10.1074/jbc.m804060200

Figure Lengend Snippet: FIGURE 2. The C-terminal -helix of yeast Alg13 is required for viability and for interaction with Alg14. A, C-terminal sequence of yeast Alg13 pro- tein. The predicted -helix is boxed. B, yeast strain (XGY154) with ALG13 under control of the glucose-repressible GAL1 promoter (PGAL1) was transformed with plasmids containing FLAG-ALG13 (pXG211), FLAG-alg13-C3 (pSA4), or FLAG-alg13-C15 (pSA6), and streaked onto YPA plates supplemented with galactose (right panel) or glucose (left panel). Cells were incubated for 2 days at 30 °C. C, whole cell detergent extracts were prepared from a wild-type strain (W303a) that co-expresses HA-ALG14 (pXG202) and FLAG-alg13-C15 (pSA6) or FLAG-ALG13 (pXG211). Samples were immunoprecipitated with anti-HA affinity matrix (lanes 1–3) or quantitatively analyzed for the presence of Alg13 and Alg13-C15 proteins by Western blotting (lanes 4–6). Proteins or extracts were separated by 12% SDS-PAGE, immunoblotted with rabbit anti-FLAG antibodies, and detected by chemiluminescence as described under “Experimental Procedures.”

Article Snippet: To detect the FLAG-tagged proteins, the membrane was blotted with a rabbit anti-FLAG polyclonal antibody (Rockland, Inc.) followed by a secondary anti-rabbit antibody conjugated to alkaline phosphatase (Chemicom, International).

Techniques: Sequencing, Control, Transformation Assay, Incubation, Immunoprecipitation, Western Blot, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

doi: 10.1074/jbc.m804060200

Figure Lengend Snippet: FIGURE 3. The C-terminal amino acids of Alg14 are required for viability and interaction with Alg13. A, C-terminal sequence of yeast Alg14 protein. The predicted -strand region is boxed. B, yeast strain (XGY151) with ALG14 under control of the glucose-repressible GAL1 promoter (PGAL1) was trans- formed with plasmids containing HA-ALG14 (pXG202), HA-alg14-C3 (pSA7), or HA-alg14-C12 (pSA8), and streaked onto YPA plates supplemented with galactose (right panel) or glucose (left panel). Cells were incubated for 2 days at 30 °C. C, a strain that contains a C-terminal triple FLAG-tagged ALG13 at the chromosomal ALG13 locus (XGY155) was transformed with plasmids encod- ing HA-ALG14 (pXG202) or HA-alg14-C3 (pSA7) or HA-alg14-C12 (pSA8). ER-enriched membrane fractions (P20) from the transformed cells were pre- pared as described under “Experimental Procedures.” Protein (80 g) from eachsamplewasseparatedby12%SDS-PAGEandquantitativelyanalyzedfor HA-tagged Alg14 proteins by immunoblotting with a mouse anti-HA mono- clonal antibody conjugated to peroxidase. D, whole cell detergent extracts were prepared from same strains used in the Western blotting assay described above. Samples were immunoprecipitated with anti-HA affinity matrix (lanes 1–4) or directly analyzed for the presence of triple FLAG-tagged Alg13p by Western blot (lanes 5–8). Immunoprecipitated proteins or extracts were separated by 12% SDS-PAGE, immunoblotted with rabbit anti-FLAG antibodies, and detected by chemiluminescence as described under “Exper- imental Procedures.”

Article Snippet: To detect the FLAG-tagged proteins, the membrane was blotted with a rabbit anti-FLAG polyclonal antibody (Rockland, Inc.) followed by a secondary anti-rabbit antibody conjugated to alkaline phosphatase (Chemicom, International).

Techniques: Sequencing, Control, Incubation, Transformation Assay, Membrane, Western Blot, Immunoprecipitation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

doi: 10.1074/jbc.m804060200

Figure Lengend Snippet: FIGURE 7. The N-terminal -strand of yeast Alg13 is required for viabil- ity and protein stability. A, N-terminal sequence of yeast Alg13 protein. The predicted -strand region is boxed. B, the PGAL1-ALG13 strain (XGY154) was transformed with plasmids containing ALG13-FLAG (pXG208), alg13- N6-FLAG (pSA1), or alg13-N10-FLAG (pSA2) and streaked onto YPA plates supplemented with galactose (right panel) or glucose (left panel). Cells were grown for 2 days at 30 °C. C, whole cell detergent extracts were prepared from wild-type yeast (W303a) containing plasmids that encode triple FLAG-tagged Alg13p (pXG208), alg13-N6p (pSA1), or alg13-N10p (pSA2). Equivalent amounts of protein in each sample were separated by SDS-PAGE, immunoblotted, and detected with anti-FLAG monoclonal antibody conjugated with alkaline phosphatase as described under “Experimental Procedures.”

Article Snippet: To detect the FLAG-tagged proteins, the membrane was blotted with a rabbit anti-FLAG polyclonal antibody (Rockland, Inc.) followed by a secondary anti-rabbit antibody conjugated to alkaline phosphatase (Chemicom, International).

Techniques: Sequencing, Transformation Assay, SDS Page

Journal: Biology of reproduction

Article Title: Effect of iron deficiency on placental cytokine expression and fetal growth in the pregnant rat.

doi: 10.1095/biolreprod66.2.516

Figure Lengend Snippet: FIG. 6. Immunolocalization of TNFa, TNFaR1, and leptin in late-gestation placentas (Day 21) from control and Fe-deficient rats. In control placentas, TNFa (A), TNFaR1 (C), and leptin (E) were detected in all trophoblast lineages. Relative to controls, placentas from Fe-deficient rats exhibited increased TNFa (B), TNFaR1 (D), and leptin (F). The inset shows the immunoglobulin G-negative control. Significance was achieved in specific trophoblast lineages: TNFa (TGC), TNFaR1 (TGC, lab, cytotrophoblasts: data not shown), and leptin (lab). Lab, Labyrinthine placenta; Sp, spongiotrophoblast;TGC, trophoblast giant cells. Bars 5 100 mm.

Article Snippet: All tissue sections were exposed to a nonimmune block with normal horse or rabbit serum (150 ml in 10 ml of buffer; Vector Laboratories, Ltd., Bretton, Peterborough, U.K.) and incubated with the appropriate primary antibodies as follows: Tissue sections were incubated with goat polyclonal anti-rat TNFa (final concentration, 4 mg/ml in TBS and 1% (w/v) BSA at 48C overnight; R&D Systems, Ltd., Abingdon, Oxfordshire, U.K.) followed by biotinylated rabbit anti-goat antibody (50 ml in 10 ml of TBS for 30 min at room temperature; Vector Laboratories).

Techniques: Control, Negative Control

Journal: Biology of reproduction

Article Title: Effect of iron deficiency on placental cytokine expression and fetal growth in the pregnant rat.

doi: 10.1095/biolreprod66.2.516

Figure Lengend Snippet: FIG. 7. Semiquantitation of TNFa (A), TNFaR1 (B), and leptin (C) in placentas from control (m) and Fe-deficient (□) rats. Immunostaining in- tensity was assessed on an arbitrary, four-point scale (0, no staining; 3, intense staining). One placenta from each dam (controls, n 5 8; deficient, n 5 7) was assessed for staining intensity in trophoblast giant cells (TGC), spongiotrophoblast (Sp), labyrinth (Lab), cytotrophoblasts (Cyt), and ad- herent deciduas (Dec). Values are mean 6 SEM. *Significant at the P , 0.05 level (Mann-Whitney test).

Article Snippet: All tissue sections were exposed to a nonimmune block with normal horse or rabbit serum (150 ml in 10 ml of buffer; Vector Laboratories, Ltd., Bretton, Peterborough, U.K.) and incubated with the appropriate primary antibodies as follows: Tissue sections were incubated with goat polyclonal anti-rat TNFa (final concentration, 4 mg/ml in TBS and 1% (w/v) BSA at 48C overnight; R&D Systems, Ltd., Abingdon, Oxfordshire, U.K.) followed by biotinylated rabbit anti-goat antibody (50 ml in 10 ml of TBS for 30 min at room temperature; Vector Laboratories).

Techniques: Control, Immunostaining, Staining, MANN-WHITNEY

Journal: Neural Regeneration Research

Article Title: Inhibition of inflammatory cytokines after early decompression may mediate recovery of neurological function in rats with spinal cord injury

doi: 10.4103/1673-5374.152374

Figure Lengend Snippet: Changes in TNF-α immunoreactivity in the spinal cord of rats with cerclage-induced spinal cord injury, with or without spinal decompression at 8 or 72 hours after injury. (A) Brown staining showing TNF-α immunoreactivity (immunohistochemical staining, × 400). (B) Quantification of TNF-α-immunoreactive cells in the rat spinal cord. Data are expressed as the mean ± SD ( n = 5 rats per group at each time point). * P < 0.05, vs . control group; # P < 0.05, vs . model group; † P < 0.05, vs . 8-hour spinal decompression group (one-way analysis of variance and independent-samples t -tests). TNF-α: Tumor necrosis factor alpha.

Article Snippet: The sections were dewaxed, hydrated, heated for antigen retrieval, treated with 3% H 2 O 2 for inactivating endogenous enzymes, blocked in 5% bovine serum albumin, and incubated with rabbit anti-TNF-α polyclonal antibody (1:200; Wuhan Boster Biological Engineering Co., Ltd., Wuhan, Hubei Province, China) at 4°C overnight, according to the manufacturer's instructions.

Techniques: Staining, Immunohistochemical staining, Control

Journal: Histochemistry and Cell Biology

Article Title: Impaired macrophages and failure of steroidogenesis and spermatogenesis in rat testes with cytokines deficiency induced by diacerein

doi: 10.1007/s00418-021-02023-7

Figure Lengend Snippet: Leydig cell (LC) steroidogenesis and ED2 testicular macrophage (TM) showing cytokines synthesis, and LC-TM paracrine interaction under normal condition. 1-) LH activates cAMP‐dependent protein kinase A, which mobilizes and transports cholesterol ( C ), derived from the blood stream. C is converted into 25-hydroxycholesterol (25-HC) by 25-hydroxylase (25-HX). StAR transports 25-HC to mitochondria where is metabolized into pregnenolone (P) by CYP11A1 enzyme. In smooth endoplasmic reticulum, P is metabolized into progesterone (PR) by 3β-HSD, and into androstenedione ( A ) by CYP17. Finally, A is converted into testosterone (T) by 17β-HSD, and T is released to the extracellular milieu. 2-) Via autocrine, T binds to androgen receptor (AR) in LC and is translocated to the nucleus, activating AR responsive genes, such as MIF, which is secreted and released by LC. 3-) LC expresses IL-1 receptor (IL-1R); IL-1β/IL-1R binding stimulates IL-1β secretion via autocrine signaling. 4-) IL-1α/β bound to IL-1R also stimulates StAR expression through ERK1/2 pathway, triggering the activation of StAR binding to 25-HC and subsequent steroidogenesis. 5-) The LC-produced MIF stimulates the recruitment of new monocytes/macrophages or is internalized by TM via CD74 receptor, stimulating NLRP3 complex and subsequent capase-1 (CASP-1), which converts Pro-IL-1β into active IL-1β. 6-) IL-1β released by LC binds to IL-1R in TM (autocrine signaling) and/or to IL-1R in LC (paracrine signaling). 7-) In TM, IL-1β triggers IkB-α degradation and activates NF-kB translocation to nucleus. NF-kB triggers genes expression, such as pro-IL-1β, CD163 and TNF-α. 8-) TM also transport 25-HC and/or 25-HX directly to LC, contributing to the steroidogenic activity of LC. Created using BioRender.com

Article Snippet: All sections were incubated in 2% BSA for 30 min, and incubated at 4 °C overnight with the following primary antibodies: mouse anti-CD163 monoclonal IgG antibody (RRID: AB_321966, 1:800, Bio-Rad, USA; code: MCA342R, lot:00,710); rabbit anti-TNF-α polyclonal IgG antibody (RRID: AB_10891701, 1:500, Boster Biological Technology, Pleasanton – USA; code: PA1079, lot:0101812Da45079125); rabbit anti-AR polyclonal IgG antibody (RRID: AB_633881, 1:1000, Santa Cruz Biotechnology, code: sc-13062, lot:#D0414); rabbit anti-IL-1β polyclonal IgG antibody (RRID: AB_308765, 1:400, Abcam, Cambridge, Massachusetts-USA; code: ab9722, lot:GR3175415-1), rabbit anti-StAR polyclonal IgG antibody (RRID:AB_2890634, 1:100, Abcam; USA; code: ab203193, lot:GR3192239-3) and mouse anti-17β-HSD7 polyclonal IgG antibody (RRID: AB_2891064, 1:500, Santa Cruz Biotechnology, Dallas, USA; code: sc-393936, lot:#B0414).

Techniques: Derivative Assay, Binding Assay, Expressing, Activation Assay, Produced, Translocation Assay, Activity Assay

Journal: Histochemistry and Cell Biology

Article Title: Impaired macrophages and failure of steroidogenesis and spermatogenesis in rat testes with cytokines deficiency induced by diacerein

doi: 10.1007/s00418-021-02023-7

Figure Lengend Snippet: Photomicrographs of testicular sections of animals from CG and DIAG submitted to immunohistochemistry for detection of AR ( a – d ) and TNF-α ( e and f ) and TNF-α immunofluorescence ( g and h ), AR protein levels by Western blot (i) and area of TNF-α immunofluorescence ( j ). In a , evident AR immunostaining is observed in the SC nuclei throughout the basal compartment of the ST (arrows), whereas in b , only few AR-immunostained SC nuclei are seen (arrows). In c and d , the interstitial tissue shows AR-immunolabelled LC (arrowheads); note that in d (DIAG), scarce AR-immunostained LC (arrowheads) are observed in comparison to CG. In e , strong TNF-α immunolabelling is observed in spermatocytes (arrows and inset) and elongating spermatids (asterisks). In f , note the weak immunoreaction observed either in the basal (arrows and inset) or in the adluminal (asterisks) compartments. In g , a seminiferous tubule at stages IX–XI shows evident TNF-α immunofluorescence in the cytoplasm of spermatocytes (thin arrows) and elongating spermatids (asterisks) whereas in h , weak or absent TNF-α immunolabelling is noted in these cells of a IX–XI tubule of animal from DIAG. In i , Western blot analysis of AR levels in testicular extracts shows strong band at 71KDa in CG in comparison with weak band in DIAG. Actin signal is observed in both groups. A significant decrease in AR levels optical density (OD) is observed in the animals from DIAG. In j , TNF-α immunofluorescence area per mm 2 of seminiferous epithelium at stages IX-XI is significantly reduced in DIAG in comparison with CG. Scale bars = 25 µm ( a and b ); 20 µm ( c and d ); 70 µm ( e and f ), 30 µm ( g and h ) and 13 µm (insets: e and f )

Article Snippet: All sections were incubated in 2% BSA for 30 min, and incubated at 4 °C overnight with the following primary antibodies: mouse anti-CD163 monoclonal IgG antibody (RRID: AB_321966, 1:800, Bio-Rad, USA; code: MCA342R, lot:00,710); rabbit anti-TNF-α polyclonal IgG antibody (RRID: AB_10891701, 1:500, Boster Biological Technology, Pleasanton – USA; code: PA1079, lot:0101812Da45079125); rabbit anti-AR polyclonal IgG antibody (RRID: AB_633881, 1:1000, Santa Cruz Biotechnology, code: sc-13062, lot:#D0414); rabbit anti-IL-1β polyclonal IgG antibody (RRID: AB_308765, 1:400, Abcam, Cambridge, Massachusetts-USA; code: ab9722, lot:GR3175415-1), rabbit anti-StAR polyclonal IgG antibody (RRID:AB_2890634, 1:100, Abcam; USA; code: ab203193, lot:GR3192239-3) and mouse anti-17β-HSD7 polyclonal IgG antibody (RRID: AB_2891064, 1:500, Santa Cruz Biotechnology, Dallas, USA; code: sc-393936, lot:#B0414).

Techniques: Immunohistochemistry, Immunofluorescence, Western Blot, Immunostaining, Comparison

Journal: Histochemistry and Cell Biology

Article Title: Impaired macrophages and failure of steroidogenesis and spermatogenesis in rat testes with cytokines deficiency induced by diacerein

doi: 10.1007/s00418-021-02023-7

Figure Lengend Snippet: Changes in Leydig cell (LC) and ED2 testicular macrophage (TM) induced by diacerein based on the literature and findings of this study. 1-) Diacerein inhibits NLRP3 complex and subsequent caspase-1 (CASP-1), impairing the conversion of pro-IL-1β into active IL-1β. 2-) Low IL-1β levels impairs IL autocrine action in TM and/or paracrine signaling in LC. 3-) In TM, NF-kB activation is impaired by low IL-1β levels, downregulating the expression of pro-IL-1β, CD163 and TNF-α. 4-) In LC, reduced IL-1α/β levels impair the auto induction of IL-1β via autocrine signaling and downregulate the expression of StAR via ERK1/2, impairing steroidogenesis. 5-) It is possible that low T levels downregulate MIF expression, reducing monocytes/macrophages recruitment (Created using BioRender.com)

Article Snippet: All sections were incubated in 2% BSA for 30 min, and incubated at 4 °C overnight with the following primary antibodies: mouse anti-CD163 monoclonal IgG antibody (RRID: AB_321966, 1:800, Bio-Rad, USA; code: MCA342R, lot:00,710); rabbit anti-TNF-α polyclonal IgG antibody (RRID: AB_10891701, 1:500, Boster Biological Technology, Pleasanton – USA; code: PA1079, lot:0101812Da45079125); rabbit anti-AR polyclonal IgG antibody (RRID: AB_633881, 1:1000, Santa Cruz Biotechnology, code: sc-13062, lot:#D0414); rabbit anti-IL-1β polyclonal IgG antibody (RRID: AB_308765, 1:400, Abcam, Cambridge, Massachusetts-USA; code: ab9722, lot:GR3175415-1), rabbit anti-StAR polyclonal IgG antibody (RRID:AB_2890634, 1:100, Abcam; USA; code: ab203193, lot:GR3192239-3) and mouse anti-17β-HSD7 polyclonal IgG antibody (RRID: AB_2891064, 1:500, Santa Cruz Biotechnology, Dallas, USA; code: sc-393936, lot:#B0414).

Techniques: Activation Assay, Expressing