Journal: Journal of Inflammation Research

Article Title: Electroacupuncture Inhibits the Early Neuroinflammatory Cascade Triggered by TLR2 in the Prodromal Period of PD

doi: 10.2147/JIR.S585729

Figure Lengend Snippet: The levels of DA and TH gradually decrease during the progression of pPD. ( A ) ELISA was used to detect the reduction in striatal DA content caused by different doses of 6-OHDA. ( B and C ) ICH was used to detect the changes in TH positive rate in substantia nigra caused by different doses of 6-OHDA (magnification ×200, scale bar 50 μm).(n=3; compared with the control group, *** P <0.001; compared with the 10 μg/4 μL group, ### P <0.001; compared with the 13 μg/4 μL group, ΔΔΔ P <0.001).

Article Snippet: The membrane was incubated overnight at 4°C with primary antibodies specific to TLR2 (#bs-1019R, Bioss, 1:1000), MyD88 (#bs-1047R, Bioss, 1:1000), p-NF-κB-p65 (#bs-7343R, Bioss, 1:1000), NLRP3 (#bs-41293R, Bioss, 1:1000), caspase-1 (#bsm-52441R, Bioss, 1:1000), IL-1β (#bs-0812R, Bioss, 1:1000), and GSDMD (#bs-14287R, Bioss, 1:1000), followed by incubation with an IgG secondary antibody (#bs-10900R, Bioss, 1:5000) for 1 hour in the dark.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Inflammation Research

Article Title: Electroacupuncture Inhibits the Early Neuroinflammatory Cascade Triggered by TLR2 in the Prodromal Period of PD

doi: 10.2147/JIR.S585729

Figure Lengend Snippet: EA can inhibit the activation of microglia in the pPD stage mediated by TLR2. ( A and B ) Immunofluorescence co-localization was used to detect the co-localization area of TLR2 in substantia nigra and the surface marker Iba-1 of microglia (magnification ×200, scale bar 50 μm). ( C ) HE staining of substantia nigra, yellow arrows indicate neuronal necrosis, and black arrows indicate inflammatory infiltration.(n=3; compared with the control group, *** P <0.001; compared with the model group, ### P <0.001).

Article Snippet: The membrane was incubated overnight at 4°C with primary antibodies specific to TLR2 (#bs-1019R, Bioss, 1:1000), MyD88 (#bs-1047R, Bioss, 1:1000), p-NF-κB-p65 (#bs-7343R, Bioss, 1:1000), NLRP3 (#bs-41293R, Bioss, 1:1000), caspase-1 (#bsm-52441R, Bioss, 1:1000), IL-1β (#bs-0812R, Bioss, 1:1000), and GSDMD (#bs-14287R, Bioss, 1:1000), followed by incubation with an IgG secondary antibody (#bs-10900R, Bioss, 1:5000) for 1 hour in the dark.

Techniques: Activation Assay, Immunofluorescence, Marker, Staining, Control

Journal: Journal of Inflammation Research

Article Title: Electroacupuncture Inhibits the Early Neuroinflammatory Cascade Triggered by TLR2 in the Prodromal Period of PD

doi: 10.2147/JIR.S585729

Figure Lengend Snippet: EA can inhibit the activation of microglia in the pPD stage mediated by TLR2. ( A ) Western blot detection of TLR2 protein expression in the substantia nigra. ( B ) Western blot detection of MyD88 protein expression in the substantia nigra. ( C ) Western blot detection of p-NF-κB-p65 expression in the substantia nigra. ( D ) Western blot detection of NLRP3 protein expression in the substantia nigra. ( E ) Western blot detection of Caspase-1 protein expression in the substantia nigra. ( F ) Western blot detection of GSDMD protein expression in the substantia nigra. ( G ) Western blot detection of IL-1β protein expression in the substantia nigra. (n=6; compared with the control group, *** P <0.001; compared with the model group, ### P <0.001, ## P <0.01, # P <0.05).

Article Snippet: The membrane was incubated overnight at 4°C with primary antibodies specific to TLR2 (#bs-1019R, Bioss, 1:1000), MyD88 (#bs-1047R, Bioss, 1:1000), p-NF-κB-p65 (#bs-7343R, Bioss, 1:1000), NLRP3 (#bs-41293R, Bioss, 1:1000), caspase-1 (#bsm-52441R, Bioss, 1:1000), IL-1β (#bs-0812R, Bioss, 1:1000), and GSDMD (#bs-14287R, Bioss, 1:1000), followed by incubation with an IgG secondary antibody (#bs-10900R, Bioss, 1:5000) for 1 hour in the dark.

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: Journal of Inflammation Research

Article Title: Electroacupuncture Inhibits the Early Neuroinflammatory Cascade Triggered by TLR2 in the Prodromal Period of PD

doi: 10.2147/JIR.S585729

Figure Lengend Snippet: EA inhibits microglial pyroptosis in the pPD. ( A ) qRT-PCR was used to detect NLRP3 mRNA in the substantia nigra. ( B ) qRT-PCR was used to detect GSDMD mRNA in the substantia nigra. ( C and D ) Co-localization of Iba-1 and GSDMD in the substantia nigra was detected by immunofluorescence co-localization (magnification ×200, scale bar 50 μm). (n=3; compared with the control group, *** P <0.001; compared with the model group, ### P <0.001, ## P <0.01).

Article Snippet: The membrane was incubated overnight at 4°C with primary antibodies specific to TLR2 (#bs-1019R, Bioss, 1:1000), MyD88 (#bs-1047R, Bioss, 1:1000), p-NF-κB-p65 (#bs-7343R, Bioss, 1:1000), NLRP3 (#bs-41293R, Bioss, 1:1000), caspase-1 (#bsm-52441R, Bioss, 1:1000), IL-1β (#bs-0812R, Bioss, 1:1000), and GSDMD (#bs-14287R, Bioss, 1:1000), followed by incubation with an IgG secondary antibody (#bs-10900R, Bioss, 1:5000) for 1 hour in the dark.

Techniques: Quantitative RT-PCR, Immunofluorescence, Control

Journal: Nucleic Acids Research

Article Title: METTL1 interacts with XPO5 to modulate pre-miRNA export

doi: 10.1093/nar/gkag037

Figure Lengend Snippet: APEX-based proximity labeling led to the identification of XPO5 as an interaction partner of METTL1. ( A ) A schematic illustration of the APEX labeling workflow. ( B ) Western blot analysis showing comparable APEX2 labeling efficiencies among METTL1-APEX, EGFP-APEX, and APEX-NLS. HEK293T cells were transfected with the respective plasmids for 24 h, treated with biotin phenol for 30 min, and subsequently exposed to H 2 O 2 for 1 min or left untreated. Biotinylated proteins were detected using streptavidin-horseradish peroxidase (SA-HRP). α-tubulin was used as a control to confirm equal protein loading. ( C ) Venn diagrams of proteins identified in the METTL1 proximal proteome. ( D ) Co-IP followed by western blot analysis in HEK293T cells showed a preferential interaction between overexpressed Flag-tagged METTL1 and endogenous XPO5. HEK293T cells were transfected with empty vector or Flag-tagged METTL1, followed by anti-Flag pull-down and western blot analysis. α-tubulin served as a loading control. XPO5 enrichment was quantified by normalizing the amount of immunoprecipitated XPO5 to its corresponding input level (mean ± SEM, *** P < .0001, unpaired t -test, n = 3).

Article Snippet: Antibodies recognizing human XPO5 (Proteintech, #28628-1-AP; 1:2000), V5 (Proteintech, #14440-1-AP; 1:2000), streptavidin (Thermo Scientific, #S911), METTL1 (Proteintech, #14994-1-AP; 1:1000), ERK (Santa Cruz, SC-514302; 1:100), and p-ERK (Cell Signaling, #4370T; 1:1000) were used as primary antibodies for western blot analysis.

Techniques: Labeling, Western Blot, Transfection, Control, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: METTL1 interacts with XPO5 to modulate pre-miRNA export

doi: 10.1093/nar/gkag037

Figure Lengend Snippet: METTL1 promotes nuclear retention of XPO5 through promoting ERK activation. ( A ) Western blot illustrating the XPO5 expression levels in the whole-cell lysates (WCL), as well as the cytoplasmic and nuclear fractions of control and METTL1 knockout cells. The result demonstrates a significant increase in cytosolic XPO5 and a significant decrease in nuclear XPO5 in METTL1 − /− cells, whereas total XPO5 remains unchanged. α-tubulin and GAPDH were used as loading controls for cytosolic fraction, and Lamin B1 as a loading control for the nuclear fraction. ( B ) Western blot analysis of ERK and p-ERK in HEK293T and METTL1 − /− cells. While total ERK expression remained unchanged, p-ERK levels were significantly reduced in the absence of METTL1. ( C ) Co-IP followed by western blot analysis showing the interaction between METTL1 and p-ERK. The P values were calculated using paired, two-tailed Student’s t -test: ns, not significant ( P > .05); *, .01 ≤ P < .05; **, .001 ≤ P < .01.

Article Snippet: Antibodies recognizing human XPO5 (Proteintech, #28628-1-AP; 1:2000), V5 (Proteintech, #14440-1-AP; 1:2000), streptavidin (Thermo Scientific, #S911), METTL1 (Proteintech, #14994-1-AP; 1:1000), ERK (Santa Cruz, SC-514302; 1:100), and p-ERK (Cell Signaling, #4370T; 1:1000) were used as primary antibodies for western blot analysis.

Techniques: Activation Assay, Western Blot, Expressing, Control, Knock-Out, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: METTL1 interacts with XPO5 to modulate pre-miRNA export

doi: 10.1093/nar/gkag037

Figure Lengend Snippet: ERK activation restores XPO5’s nuclear retention in METTL1 −/− cells. ( A ) Western blot analysis of nuclear XPO5 in METTL1 −/− cells transfected with Flag-MEK1 or Flag-MEKDD. ( B ) Western blot analysis showing p-ERK and XPO5 in HEK293T (labeled as “Ctrl”) and the isogenic METTL1 −/− cells. GAPDH was used as a loading control. ( C ) Subcellular fractionation analysis of XPO5 in cytosolic and nuclear compartments in HEK293T and METTL1 −/− cells expressing Flag-MEKDD. GAPDH and Lamin B1 were used as loading controls for the cytosolic and nuclear fractions, respectively. The P -values were calculated using a paired, two-tailed Student’s t -test: ns, not significant ( P > .05); *, .01 ≤ P < .05.

Article Snippet: Antibodies recognizing human XPO5 (Proteintech, #28628-1-AP; 1:2000), V5 (Proteintech, #14440-1-AP; 1:2000), streptavidin (Thermo Scientific, #S911), METTL1 (Proteintech, #14994-1-AP; 1:1000), ERK (Santa Cruz, SC-514302; 1:100), and p-ERK (Cell Signaling, #4370T; 1:1000) were used as primary antibodies for western blot analysis.

Techniques: Activation Assay, Western Blot, Transfection, Labeling, Control, Fractionation, Expressing, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: DHCR24 Deficiency Drives Age-Related Meibomian Gland Dysfunction by Regulating Lipid Metabolic Imbalance and Cytosolic mtDNA-Induced cGAS-STING Activation

doi: 10.7150/ijbs.129636

Figure Lengend Snippet: Activation of cytokines and inflammatory pathways in meibocyte-specific Dhcr24 conditional knockout (cKO) mice. ( A - C ) Gene ontology (GO) analysis of differentially expressed genes (DEGs) revealing significantly enriched pathways in three categories, including ( A ) biological process (BP), ( B ) cellular component (CC), and ( C ) molecular function (MF). ( D ) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of DEGs between Dhcr24 fl/fl and Dhcr24 -cKO mice. ( E ) Representative immunofluorescence (IF) images of interleukin-6 (IL-6), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α) in Dhcr24 -cKO vs. Dhcr24 fl/fl mice. Scale bars: 50 μm. ( F-H ) Enzyme-linked immunosorbent assay (ELISA) measurement of meibomian gland IL-6, IL-17, and TNF-α concentrations from Dhcr24 -cKO and Dhcr24 fl/fl mice. ( I ) Representative IF images of nuclear factor κB (NF-κB) p65 and phospho-NF-κB p65 in Dhcr24 -cKO vs. Dhcr24 fl/fl group. Scale bars: 50 μm. ( J , K ) Heatmap based on differentially expressed genes for significantly enriched pathways of ( J ) cytokines and inflammatory reponse and ( K ) NF-κB signaling. ( L ) Western blot of phospho-NF-κB p65 and NF-κB p65 protein levels. ( M ) Relative fluorescence intensity and ( N ) protein levels of phospho-NF-κB p65 and NF-κB p65.

Article Snippet: Mouse MG cryosections were permeabilized (0.1% Triton X-100), blocked with 5% BSA, and incubated with primary antibodies against KRT14 (Bioss; #bsm-52054R), DHCR24 (Bioss; #bs-5390R), IL-6 (Proteintech; 21865-1-AP), IL-17 (Bioss; #bs-1183R), TNF-α (Proteintech; #17590-1-AP), NF-κB p65 (Abcam; #ab32536), phospho-NF-kB p65 (S536) (Abcam; #ab76302), and STING (Proteintech; #19851-1-AP) overnight.

Techniques: Activation Assay, Knock-Out, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: DHCR24 Deficiency Drives Age-Related Meibomian Gland Dysfunction by Regulating Lipid Metabolic Imbalance and Cytosolic mtDNA-Induced cGAS-STING Activation

doi: 10.7150/ijbs.129636

Figure Lengend Snippet: Adeno-associated virus (AAV)-mediated DHCR24 overexpression reverses ocular surface pathology in Dhcr24 conditional knockout (cKO) mice. ( A ) Experiment timeline for AAV treatment and ocular surface assessments. Two-month-old Dhcr24 -cKO and Dhcr24 fl/fl mice were injected with tamoxifen (TAM) for 7 days and raised for 2 weeks before intra-meibomian gland (MG) injection of AAV-vehicle or AAV-DHCR24, followed by a 3-month period before ocular phenotype evaluation. A separate group of two-month-old Dhcr24 -cKO mice was raised for 6 months post-TAM before undergoing the same AAV treatment and evaluation procedures. ( B ) Representative white light photos, fluorescein staining, Rose Bengal staining, and upper tarsal plate images for Dhcr24 fl/fl , young Dhcr24 -cKO (3m), and aged Dhcr24 -cKO (9m) with AAV-vehicle or AAV-DHCR24 treatment. Arrows pointing to truncated or absent MGs. ( C - F ) Ocular surface and MG scoring in the treatment groups, including ( C ) corneal opacity score ( n = 5), ( D ) corneal fluorescein staining score ( n = 5), ( E ) Rose Bengal staining score ( n = 5), and ( F ) clinical MG score ( n = 5). ( G ) Oil Red O staining of upper tarsal plate sections in the different treatment groups, with circles indicating absent MGs. Scale bars: 200 μm. ( H ) Hematoxylin-eosin (H&E) staining comparing the Dhcr24 -cKO mice treated with AAV-vehicle and AAV-DHCR24. Scale bars: 50 μm. ( I ) Immunofluorescence (IF) staining and ( J ) relative fluorescence intensity of STING for young Dhcr24 -cKO (3m) and aged Dhcr24 -cKO (9m) treated with either AAV-vehicle or AAV-DHCR24. Scale bars: 50 μm. ( K-M ) Relative mRNA expression of ( K ) interleukin-6 (IL-6), ( L ) interleukin-17 (IL-17), and ( M ) tumor necrosis factor-α (TNF-α) in mouse MGs of Dhcr24 -cKO (3m) and Dhcr24 -cKO (9m) following AAV-vehicle or AAV-DHCR24 treatment.

Article Snippet: Mouse MG cryosections were permeabilized (0.1% Triton X-100), blocked with 5% BSA, and incubated with primary antibodies against KRT14 (Bioss; #bsm-52054R), DHCR24 (Bioss; #bs-5390R), IL-6 (Proteintech; 21865-1-AP), IL-17 (Bioss; #bs-1183R), TNF-α (Proteintech; #17590-1-AP), NF-κB p65 (Abcam; #ab32536), phospho-NF-kB p65 (S536) (Abcam; #ab76302), and STING (Proteintech; #19851-1-AP) overnight.

Techniques: Virus, Over Expression, Knock-Out, Injection, Staining, Immunofluorescence, Fluorescence, Expressing