Journal: Redox Report : Communications in Free Radical Research

Article Title: The novel thioredoxin reductase inhibitor butaselen suppresses lung cancer by inducing oxidative stress

doi: 10.1080/13510002.2025.2588086

Figure Lengend Snippet: BS affects NF-κB, p38/JNK, and PI3K-Akt signaling pathways in lung cancer. (a–d) A549 and H1299 cells were treated with BS (0, 5, 10 μM) for 24 h, followed by western blot. Error bars are means ± std. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the control group by One-way ANOVA (n = 3).

Article Snippet: The primary antibodies used for immunoblotting analysis are as follows: TrxR1 (Proteintech, 11117-1-AP), Trx1 (Proteintech, 14999-1-AP), HBP1 (Proteintech, 11746-1-AP), DNMT1 (Proteintech, 24206-1-AP), Bcl-2 (Proteintech, 12789-1-AP), Bax (Proteintech, 50599-2-Ig), β-actin (Bioss, bs-0061R), Flag (Sigma-Aldrich, F1804), HA (Covance, MMS-101P), p53 (Santa, sc-126), p21 (MBL, K0081-3), p27 (MBL, K0082), γ-H2AX (CST, 9718), NF-κB (Abcam, ab32536), p-NF-κB(CST, 3033), p38 (Santa, sc-7972), p-p38 (Santa, sc-101759), JNK (MCE, HY- P80728 ), p-JNK (Immunoway, YP0157), Akt (Santa, sc-5298), phospho-Akt (CST, 4060).

Techniques: Protein-Protein interactions, Western Blot, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: The novel thioredoxin reductase inhibitor butaselen suppresses lung cancer by inducing oxidative stress

doi: 10.1080/13510002.2025.2588086

Figure Lengend Snippet: Schematic model of lung cancer inhibition by BS. The TrxR/Trx inhibitor butaselen (BS) can inhibit lung cancer by inducing ROS-dependent apoptosis. The inactivation of the NF-κB and PI3K-Akt signaling pathways, along with the activation of the p38/JNK signaling pathway, contributes to the anti-cancer effects of BS on lung cancer. Although p53 itself is not activated by BS, the HBP1/DNMT1/p21/γ-H2AX/Bcl-2/Bax signaling pathway is activated by BS and contributes to its tumor-inhibitory role. Further mechanistic studies revealed HBP1 as a novel target of the Trx system. The Trx system inversely associates with HBP1 in lung cancer and regulates HBP1 expression at the post-translational level. Under normal conditions, TrxR1 catalyzes the reduction of Trx1 by utilizing NADPH. In its reduced form, Trx1 interacts with HBP1, promoting the ubiquitination of HBP1, which leads to its proteasomal degradation and maintains a low level of HBP1 within cancer cells. Treatment with butaselen inhibits the activity of TrxR1 in lung cancer cells, resulting in the oxidation of Trx1 and the subsequent excessive generation of ROS. HBP1 is activated after being released by the oxidized Trx1 and escaping proteasomal degradation. The activated HBP1 inhibits the expression of DNMT1 and elevates Bax. The decreased DNMT1 further results in the demethylation of the whole genome as well as the promoters of p21 and HOXA9. Ultimately, the upregulation of p21 and γ-H2AX, along with the downregulation of DNMT1 and Bcl-2/Bax, contributes to the apoptosis of lung cancer cells induced by BS. Taken together, the TrxR/Trx inhibitor butaselen inhibits lung cancer by promoting ROS-induced apoptosis through the NF-κB, PI3K-Akt, p38/JNK, and HBP1/DNMT1 signaling pathways.

Article Snippet: The primary antibodies used for immunoblotting analysis are as follows: TrxR1 (Proteintech, 11117-1-AP), Trx1 (Proteintech, 14999-1-AP), HBP1 (Proteintech, 11746-1-AP), DNMT1 (Proteintech, 24206-1-AP), Bcl-2 (Proteintech, 12789-1-AP), Bax (Proteintech, 50599-2-Ig), β-actin (Bioss, bs-0061R), Flag (Sigma-Aldrich, F1804), HA (Covance, MMS-101P), p53 (Santa, sc-126), p21 (MBL, K0081-3), p27 (MBL, K0082), γ-H2AX (CST, 9718), NF-κB (Abcam, ab32536), p-NF-κB(CST, 3033), p38 (Santa, sc-7972), p-p38 (Santa, sc-101759), JNK (MCE, HY- P80728 ), p-JNK (Immunoway, YP0157), Akt (Santa, sc-5298), phospho-Akt (CST, 4060).

Techniques: Inhibition, Protein-Protein interactions, Activation Assay, Expressing, Ubiquitin Proteomics, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 8. Decreased proliferation and increased chemokine production of epidermal keratinocytes from miR-132–KO mice. (A) H&E staining of the skin from miR-132–KO mice (n = 9) and their WT littermate controls (n = 5) and epidermis thickness were analyzed. Scale bars: 100 μm. (B) Immunostaining of Ki-67 and HB-EGF in skin from WT (n = 9) and KO mice (n = 5). Sections were counterstained using DAPI (blue, nucleus). The number of Ki-67–positive cells was counted. Scale bars: 50 μm. (C) Epidermal keratinocytes were isolated from the skin of WT (n = 3) and KO (n = 3) mice. Ccl20, Cxcl5, Cxcl1, Tnf, Il1a, Il1b, and Hbegf expression levels were analyzed using qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Staining, Immunostaining, Isolation, Expressing, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 9. Skin wound healing is impaired in miR-132–KO mice. (A) Representative images of wounds on the backs of miR-132–KO mice (n = 9) and their WT littermate controls (n = 6) on days 0, 2, 4, 6, and 8 after wounding. Wound closures were quantified and are presented as the percentage of the initial wound area size. (B) qRT-PCR was performed to detect Ccl20, Cxcl5, Cxcl1, Tnf, Il1a, and Il1b expression levels in the intact and wound-edge skin of both WT (n = 5) and KO (n = 9) mice 2 days after injury. *P < 0.05 and **P < 0.01 by Student’s t test. (C) H&E staining (left panels) and immunostaining of Gr-1 (mid- dle and right panels) in the wound area in WT and KO mice 2 days after injury. White arrows demarcate wound edges. Scale bars: 50 μm. (D) Chemotaxis of neutrophils isolated from the peripheral blood of WT (n = 5) and KO mice (n = 7) toward the medium containing 8 μM N-formylmethionyl-leucyl-phenyla- lanine (fMLP). Migrating cells were quantified by flow cytometry. Plots show the FSC/SSC of the migrated cells. (E) Immunostaining of Ki-67 and HB-EGF in wound-edge skin from WT (n = 9) and KO mice (n = 5) 2 days after injury. Sections were counterstained using DAPI (blue, nucleus). The number of Ki-67– positive cells was counted. (F) Immunostaining of p-STAT3 and p-ERK in wound-edge skin from WT (n = 9) and KO mice (n = 5) 2 days after injury. Scale bars: 50 μm (E and F). *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Staining, Immunostaining, Chemotaxis Assay, Isolation, Flow Cytometry

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 11. Inhibition of miR-132 delays reepithelialization of human ex vivo skin wounds. Human ex vivo skin wounds were treated topically with anti–miR-132 (n = 4) or anti–miR-Ctrl (n = 4) after injury, and miR-132 blocking efficiency was confirmed by qRT-PCR (A). (B) H&E staining of the ex vivo skin wounds 3 days after injury. Black arrows demarcate the initial wound edges, while the red arrows indicate a newly formed epidermis. Scale bars: 200 μm. (C) Immunostaining of Ki-67 in ex vivo skin wounds 3 days after injury. Sections were counterstained with DAPI. The number of Ki-67– positive cells was counted. White arrows demarcate the wound edges. Scale bars: 50 μm. CXCL1, CXCL5, CCL20 (D), and HB-EGF (E) expression levels were detected by qRT-PCR in ex vivo wounds treated with anti–miR-Ctrl (n = 4) or anti–miR-132 (n = 4) 3 days after injury. (F) Schematic summary of the regulation and function of miR-132 during skin wound healing. *P < 0.05 and **P < 0.01 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Inhibition, Ex Vivo, Blocking Assay, Quantitative RT-PCR, Staining, Immunostaining, Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Hawthorne leaf flavonoids prevent oxidative stress injury of renal tissues in rats with diabetic kidney disease by regulating the p38 MAPK signaling pathway

doi:

Figure Lengend Snippet: Immunochemical analysis of the (A) p38MAPK (B) and p-p38MAPK expression in the kidney of different groups (200×). Quantitative analysis of (C) p38MAPK and (D) p-p38MAPK expression in the kidney of different groups. The expression of p38MPAK and p-p38MAPK was significantly higher in the DKD group than in the HLF and IRB groups.

Article Snippet: Rabbit anti-rat p38MAPK and p-p38MAPK polyclonal antibodies were purchased from Boster (Beijing, China).

Techniques: Expressing

Journal: Molecular medicine reports

Article Title: Placental growth factor gene silencing mitigates the epithelial‑to‑mesenchymal transition via the p38 MAPK pathway in rats with hyperoxia‑induced lung injury.

doi: 10.3892/mmr.2019.10785

Figure Lengend Snippet: Figure 5. Expression of p38 and p‑p38 MAPK in hyperoxia‑exposed lung tissue. Neonatal rats were exposed to normoxic or hyperoxic conditions for 14 days; hyperoxia exposed rats were subsequently injected with shRNA‑NC or shRNA‑PLGF. (A) Western blot analysis and (B) quantification was performed to determine p‑p38/p38 protein expression levels in rat lung tissues; β‑actin was used as the endogenous control. Data are presented as the mean ± standard deviation. n=8. ***P<0.001. MAPK, mitogen‑activated protein kinase; NC, negative control; PLGF, placental growth factor; shRNA, short hairpin RNA.

Article Snippet: The animals were housed at a temperature of 25‐27 ̊C, with a humidity of 50-70% and a 12 h light/dark cycle with ad libitum access to food and water. xylene, absolute ethanol, eosin y and hydrogen peroxide were purchased from Wuhan uScn Business co., ltd. Hematoxylin, eosin and goat serum (cat. no. Sl038) were purchased from Beijing Solarbio Science& Technology co., ltd. PlGF mouse monoclonal antibody (cat. no. sc-518003) and e-cadherin mouse monoclonal antibody (cat. no. sc-71007) were purchased from Santa cruz Biotechnology, inc. anti-phosphorylated (p)-p38 rabbit polyclonal antibody (cat. no. bs-2210r) was purchased from (BioSS). anti-p38 rabbit monoclonal (cat. no. M00176), anti-β-actin goat polyclonal (cat. no. BM0627) and anti-ZeB2 rabbit polyclonal (cat. no. Pa1959) antibodies were purchased from Boster Biological Technology.

Techniques: Expressing, Injection, Western Blot, Control, Standard Deviation, Negative Control, shRNA

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Myrica rubra Extracts Protect the Liver from CCl 4 -Induced Damage

doi: 10.1093/ecam/nep196

Figure Lengend Snippet: Effect of MCE on CCl 4 -induced acute liver damage in mice.

Article Snippet: Et Zucc. extract (MCE) against liver injury induced by CCl 4 , addressing the possible action of MCE on liver mitochondrial and VDAC expression in order to search for the mechanism underlying its hepatoprotective activity.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Myrica rubra Extracts Protect the Liver from CCl 4 -Induced Damage

doi: 10.1093/ecam/nep196

Figure Lengend Snippet: Photomicrographs of liver sections taken from mice treated with CCl 4 with or without pretreatment with MCE. (a) Normal; (b) CCl 4 ; (c) CCl 4 + MCE L ; (d) CCl 4 + MCE M ; and (e) CCl 4 + MCE H .

Article Snippet: Et Zucc. extract (MCE) against liver injury induced by CCl 4 , addressing the possible action of MCE on liver mitochondrial and VDAC expression in order to search for the mechanism underlying its hepatoprotective activity.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Myrica rubra Extracts Protect the Liver from CCl 4 -Induced Damage

doi: 10.1093/ecam/nep196

Figure Lengend Snippet: Protective effect of MCE on the ultrastructure of hepatocytes induced by CCl 4 . Mice were divided into five groups: (a) Normal, (b) CCl 4 , (c) CCl 4 + MCE L , (d) CCl 4 + MCE M and (e) CCl 4 + MCE H . The CCl 4 and different MCE groups were administered saline or various concentrations of MCE orally for 5 days before the intra-peritoneal injection of 0.30% CCl 4 . Specimens were taken 24 h later and regularly prepared for examination under an electron microscope (×5000).

Article Snippet: Et Zucc. extract (MCE) against liver injury induced by CCl 4 , addressing the possible action of MCE on liver mitochondrial and VDAC expression in order to search for the mechanism underlying its hepatoprotective activity.

Techniques: Saline, Injection, Microscopy

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Myrica rubra Extracts Protect the Liver from CCl 4 -Induced Damage

doi: 10.1093/ecam/nep196

Figure Lengend Snippet: Prevention of MCE on mitochondrial membrane potential dissipation induced by CCl 4 . Mice were treated with MCE for 5 days before pretreatment with CCl 4 . Liver mitochondria were isolated and the mitochondrial membrane potential was determined using Rh123. Each value represents mean ± SD ( n = 8). * P < .05, ** P < .01, versus Normal; + P < .05 versus CCl 4 group.

Article Snippet: Et Zucc. extract (MCE) against liver injury induced by CCl 4 , addressing the possible action of MCE on liver mitochondrial and VDAC expression in order to search for the mechanism underlying its hepatoprotective activity.

Techniques: Membrane, Isolation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Myrica rubra Extracts Protect the Liver from CCl 4 -Induced Damage

doi: 10.1093/ecam/nep196

Figure Lengend Snippet: Effect of MCE on liver mitochondrial calcium content in mice treated with CCl 4 . Mice were treated with MCE for 5 days before pretreatment with CCl 4 . Liver mitochondria were then isolated and mitochondrial free calcium content was determined using Fluo-3. MCE showed a dose-dependent suppression of the CCl 4 -induced intra-mitochondrial Ca 2+ overload. Values represent mean ± SD ( n = 6). * P < .01 versus the normal group; + P < .01 versus the CCl 4 group.

Article Snippet: Et Zucc. extract (MCE) against liver injury induced by CCl 4 , addressing the possible action of MCE on liver mitochondrial and VDAC expression in order to search for the mechanism underlying its hepatoprotective activity.

Techniques: Isolation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Myrica rubra Extracts Protect the Liver from CCl 4 -Induced Damage

doi: 10.1093/ecam/nep196

Figure Lengend Snippet: Effect of MCE on Ca 2+ -induced mitochondrial swelling. CCl 4 can decrease the sensitivity in Ca 2+ -induced mitochondrial swelling. MCE dose-dependently recuperated this sensitivity. The curves represent typical recordings from experiments of at least three different mitochondrial preparations.

Article Snippet: Et Zucc. extract (MCE) against liver injury induced by CCl 4 , addressing the possible action of MCE on liver mitochondrial and VDAC expression in order to search for the mechanism underlying its hepatoprotective activity.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Myrica rubra Extracts Protect the Liver from CCl 4 -Induced Damage

doi: 10.1093/ecam/nep196

Figure Lengend Snippet: Effect of MCE on mitochondrial VDAC expression in CCl 4 -insulted mouse livers. Livers from various groups were taken 24 h following 0.30% CCl 4 . (a) Inhibitory effect of MCE on the decrease in VDAC mRNA level induced by CCl 4 analyzed by RT-PCR. (b) Inhibitory effect of MCE on the decrease in VDAC protein level induced by CCl 4 analyzed by western blot.

Article Snippet: Et Zucc. extract (MCE) against liver injury induced by CCl 4 , addressing the possible action of MCE on liver mitochondrial and VDAC expression in order to search for the mechanism underlying its hepatoprotective activity.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Myrica rubra Extracts Protect the Liver from CCl 4 -Induced Damage

doi: 10.1093/ecam/nep196

Figure Lengend Snippet: CCl 4 could induce peroxidation of the unsaturated fatty acids of cell membrane, and lead to membrane injury, enhance the leakage of AST, the dissipation of mitochondrial membrane potential, hepatocellular Ca 2+ overload and decrease the sensitivity in Ca 2+ -induced mitochondrial swelling and other the mRNA and the protein of VDAC's expressions. Using MCE could prevent all these changes, that is to say, MCE has hepatoprotective activity and the mechanisms underlying its protective effects may be related to the mitochondrial protection.

Article Snippet: Et Zucc. extract (MCE) against liver injury induced by CCl 4 , addressing the possible action of MCE on liver mitochondrial and VDAC expression in order to search for the mechanism underlying its hepatoprotective activity.

Techniques: Membrane, Activity Assay

Journal: Frontiers in pharmacology

Article Title: Neuroprotective Effects and Mechanisms of Zhenlong Xingnao Capsule in In Vivo and In Vitro Models of Hypoxia.

doi: 10.3389/fphar.2019.01096

Figure Lengend Snippet: FIGURE 6 | Effects of ZXC on the mRNA levels of the Bcl-2/Bax ratio (A), caspase-3 (B), nuclear factor (NF)-кB (C), and p38 (D) in the prefrontal cortex of ischemia-reperfusion injury rats. The data are expressed as mean ± standard deviation (n = 3). I-30, ischemia for 30 min; I-90, ischemia for 90 min; I-90+R-30, ischemia for 90 min, then reperfusion for 30 min; I-90+R-180, ischemia for 90 min, then reperfusion for 180 min. *P < 0.05 vs. sham group; #P < 0.05 vs. model group; ##P < 0.01 vs. model group.

Article Snippet: The primary antibodies used for IHC were the MAPK14 (p38) antibody and CASP3 (P17) antibody; both were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Standard Deviation

Journal: Frontiers in pharmacology

Article Title: Neuroprotective Effects and Mechanisms of Zhenlong Xingnao Capsule in In Vivo and In Vitro Models of Hypoxia.

doi: 10.3389/fphar.2019.01096

Figure Lengend Snippet: FIGURE 7 | Western blotting results for Sham, Model and ZXC groups (A). Effects of ZXC on the protein expressions Bcl-2 (B), Bax (C), Caspase-3 (D), NF-кB (E), and p38 (F) in brain tissue of ischemia-reperfusion injury rats induced by MCAO. The data are expressed as mean ± standard deviation (n = 4). I-30, ischemia for 30 min; I-90, ischemia for 90 min; I-90+R-30, ischemia for 90 min, then reperfusion for 30 min. *P < 0.05 vs. sham group; #P < 0.05 vs. model group.

Article Snippet: The primary antibodies used for IHC were the MAPK14 (p38) antibody and CASP3 (P17) antibody; both were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: Mixed neuronal-glial cerebrocortical cultures from WT or IFNAR1KO mice were incubated with IFNβ in increasing doses (from 500 to 5,000 U/ml) or BSA/PBS vehicle control for 0, 3, 6, 12 and 24 h. Total RNA was extracted from cell lysates, analyzed by qRT-PCR and normalized to GAPDH expression levels. ( a–d ) RNA expression is shown as fold change (FC) in relation to vehicle treated controls which were defined as baseline activity. ( e–h ) Time course for protein expression measured in cell-free supernatants for CCL3, CCL4, CCL5 and CXCL10 using a commercially available multiplex assay as described in Methods. Baseline protein expression in vehicle treated cell cultures is represented as 0 h time point. Values are mean ± s.e.m.; n = 3–5 independent experiments per ISG; ***p < 0.001, **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test. For clarity, the significance is only indicated for differences between treatments and baseline within each experimental group.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Incubation, Control, Quantitative RT-PCR, Expressing, RNA Expression, Activity Assay, Multiplex Assay

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: ( a ) Mixed neuronal-glial cerebrocortical cultures from WT mice were simultaneously exposed for 3 days to HIV gp120 BaL (200 pM) and mouse IFNβ (5,000 U/ml) in the presence and absence of neutralizing antibodies against CCL3, CCL4, CCL5, IFNγ or CXCL10. IFNγ antibody was used as control for neutralizing antibodies since this protein was undetectable in cerebrocortical cell cultures. ( b ) Mouse cerebrocortical cultures from IFNAR1KO mice were stimulated with gp120 BaL for 24 h the presence or absence of mouse IFNβ (5,000 U/ml) or BSA/PBS control. ( c ) Cerebrocortical cell cultures from WT mice were simultaneously exposed for 3 days to HIV gp120 BaL in the presence and absence of murine CCL4 (2 or 20 nM). Neuronal survival was assessed by immunofluorescence microscopy and counting of MAP-2/NeuN double-positive neurons. Values are mean ± s.e.m.; n = 3–5 independent experiments with 3–7 replicates and an average of 9,000 (IFNAR1KO) or 5,700 (WT) cells counted per condition; ** p < 0.01, *** p < 0.001 by ANOVA with Fisher’s PLSD post hoc test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Control, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: RNA was purified from one brain hemisphere each of 4–5 month-old HIVgp120tg and WT littermate mice previously treated with intranasal IFNβ or vehicle and analyzed by qRT-PCR for fold-change (FC) in ISG expression. Significant changes in gene expression were observed between IFNβ and vehicle treatment groups in WT brains ( a ) for CCL4, and in gp120tg brains ( b ) for CCL4, CXCL11 and IRF3. Expression of transgenic HIVgp120 was not affected by IFNβ ( c ). Values are mean ± s.e.m.; n = 4–5 animals per group/genotype; *p < 0.05, student’s t-test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Purification, Quantitative RT-PCR, Expressing, Gene Expression, Transgenic Assay

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: Sagittal brains sections of HIVgp120tg and WT littermate mice previously treated with intranasal IFNβ or vehicle (veh) were immunolabeled for CCL4, neuronal MAP-2 or astrocytic GFAP. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was labeled with Hoechst (H) 33342. The fluorescence-labeled brain sections were analyzed using confocal laser-scanning microscopy. Representative images of cortex layer III are shown; scale bar, 50 μm.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Immunolabeling, Labeling, Fluorescence, Confocal Laser Scanning Microscopy

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: ( a ) Cerebrocortical cultures from mice were prepared to either contain microglia, neurons and astrocytes (M + N + A) or were depleted of microglia (N + A) or neurons and microglia (A). Complete and depleted cell cultures were incubated with mIFNβ (5,000 U/ml) or BSA/PBS vehicle control for 0, 3, 6, 12 and 24 h and concentrations of CCL4 were measured in cell-free supernatants using a commercially available multiplex assay as described in Methods. Maximum concentrations were reached in samples of 12 to 24 h mIFNβ exposure and compared to vehicle-treated, baseline samples. Values are mean ± s.e.m.; n = 3 independent experiments; *p < 0.05, student’s t-test. ( b ) Microglia-depleted rat cerebrocortical cultures were exposed for 24 h to 50% cell-free conditioned media (CM) from human MDM in the presence or absence of human IFNβ (5,000 U/ml). MDM were previously stimulated for 24 h with HIV-1 gp120 BaL (MDM gp120 CM) or vehicle (MDM CM). Following the incubation the cells were fixed and permeabilized. Neurons were immunolabeled for neuronal MAP-2 and NeuN and nuclear DNA was stained with H33342. Neuronal survival was assessed using fluorescence microscopy and cell counting as described in Methods. Values are mean ± s.e.m.; n = 2 independent experiments, with 4–8 replicates and an average of 4,000 cells counted per condition; **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Incubation, Control, Multiplex Assay, Immunolabeling, Staining, Fluorescence, Microscopy, Cell Counting

Journal: NPJ Science of Food

Article Title: Effects of Mytilus edulis derived plasmalogens against atherosclerosis via lipid metabolism and MAPK signaling pathway

doi: 10.1038/s41538-025-00546-0

Figure Lengend Snippet: HFD has a significant pro-atherogenic effect, which might be associated with provocation of AP-1 mediated inflammatory response mediated by calmodulin. In contrast, Pls supplementation effectively inhibits HFD-induced activation of MAPK signaling pathway, thereby preventing and the downstream inflammatory damage and blocking the development of HFD-related disorders, including atherosclerosis.

Article Snippet: Subsequently, the membranes were washed with TBST three times, and incubated with the following primary antibodies: β-actin (HY- P80438 , MedChemExpress, Monmouth Junction, NJ, USA), p38 MAPK (HY- P80776 , MedChemExpress), Casp1 (ab138483, abcam, Cambridge, UK), Calm4 (ab2860, abcam), and Tnf (HY-P7090, MedChemExpress) at 4 °C overnight with agitation, followed by incubation with the second antibody for 4 h. The protein bands were visualized using electrochemiluminescence, and the intensity of bands was quantitatively analyzed using ImageJ software, with β-actin as the protein loading control.

Techniques: Activation Assay, Blocking Assay