Journal: Investigative Ophthalmology & Visual Science

Article Title: AMPK Deficiency Induces Corneal Epithelial Barrier Dysfunction by Modulating Energy Homeostasis

doi: 10.1167/iovs.67.2.47

Figure Lengend Snippet: Construction and verification of AMPKα1α2CE-KO mice. (A) Breeding strategy. Cre recombinase excised the loxP-flanked exon 3 of AMPKα1 and exon 2 of AMPKα2; (B) Representative agarose gels showing the genotyping of wild type (WT) mice and AMPKα 1 α 2 CE-KO KO mice; PCR was performed on tail genomic DNA using primers (shown in red ), generating products of 334 bp and 341 bp from the WT allele, 450 bp from the floxed allele; (C) Representative images of immunofluorescence staining for AMPK ( red ) and K12 ( green ) in cornea of AMPKα1α2 f/f (Flox) and KO mice. Scale bar : 50 µm. (D) The immunofluorescence intensity of AMPKα in corneal epithelium. (E, F) Western blot and quantitative analysis of protein expression of p-AMPKα, AMPKα, and p-ACC in corneal epithelium with β-actin as a loading control. n = 3 (B–D) , n = 5 (E and F) . Data shown as mean ± SD. *** P < 0.001.

Article Snippet: Primary antibodies used in this study were as follows: p-AMPKα (2535, 1:1000), AMPKα (5831, 1:1000), phosphorylated acetyl-CoA carboxylase (p-ACC, 11818, 1:1000), ACC (3676, 1:1000), and Dynamin-related protein 1 (DRP1; 5391, 1:1000) from Cell Signaling Technology (Danvers, MA, USA); ZO-1 (21773-1-AP, 1:1000), Occludin (66378-1-lg, 1:1000), E-cadherin (CDH, 20874-1-AP, 1:1000), Mitofusion 2 (MFN2, 12186-1-AP, 1:1000), Optic atrophy 1 (OPA1, 27733-1-AP, 1:1000), MFF (17090-1-AP, 1:1000), IL-1β (16806-1-AP, 1:1000), IL-10 (60269-1-lg, 1:1000), Mitochondria Complex IV (MTCO2, 55070-1-AP, 1:1000), β-actin (HRP-60008, 1:10,000), and secondary antibodies (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG), all purchased from Proteintech (Wuhan, China).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: AMPK Deficiency Induces Corneal Epithelial Barrier Dysfunction by Modulating Energy Homeostasis

doi: 10.1167/iovs.67.2.47

Figure Lengend Snippet: AMPK deficiency induces disruption of epithelial AJCs in vivo and in vitro. (A, B) Western blot and quantitative analysis of protein expression of ZO1, Occludin, and CDH1 in corneal epithelium with β-actin as a loading control. (C) Representative images of immunofluorescence staining for ZO1 ( green ) in the cornea of Flox and KO mice; Scale bar : 50 µm. (D) The immunofluorescence intensity of ZO1 in corneal epithelium. (E) The mRNA expression of AMPKα1 and AMPKα2 in HCECs transfected with siCtrl and siAMPK. (F) Western blot and quantitative analysis of protein expression of AMPKα in HCECs with β-actin as a loading control. (G) Representative images of immunofluorescence staining for ZO1 ( green ) in HCECs. Scale bar : 25 µm. (H) Western blot and quantitative analysis of protein expression of ZO1, Occludin, and CDH1 in HCECs with β-actin as a loading control. n = 4. Data was shown as mean± SD. * P < 0.05, *** P < 0.001.

Article Snippet: Primary antibodies used in this study were as follows: p-AMPKα (2535, 1:1000), AMPKα (5831, 1:1000), phosphorylated acetyl-CoA carboxylase (p-ACC, 11818, 1:1000), ACC (3676, 1:1000), and Dynamin-related protein 1 (DRP1; 5391, 1:1000) from Cell Signaling Technology (Danvers, MA, USA); ZO-1 (21773-1-AP, 1:1000), Occludin (66378-1-lg, 1:1000), E-cadherin (CDH, 20874-1-AP, 1:1000), Mitofusion 2 (MFN2, 12186-1-AP, 1:1000), Optic atrophy 1 (OPA1, 27733-1-AP, 1:1000), MFF (17090-1-AP, 1:1000), IL-1β (16806-1-AP, 1:1000), IL-10 (60269-1-lg, 1:1000), Mitochondria Complex IV (MTCO2, 55070-1-AP, 1:1000), β-actin (HRP-60008, 1:10,000), and secondary antibodies (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG), all purchased from Proteintech (Wuhan, China).

Techniques: Disruption, In Vivo, In Vitro, Western Blot, Expressing, Control, Immunofluorescence, Staining, Transfection

Journal: Investigative Ophthalmology & Visual Science

Article Title: AMPK Deficiency Induces Corneal Epithelial Barrier Dysfunction by Modulating Energy Homeostasis

doi: 10.1167/iovs.67.2.47

Figure Lengend Snippet: AMPK deficiency impairs mitochondrial homeostasis in corneal epithelial cells. (A) Representative images of Rhodamine ( green ) staining in HCECs. Scale bar : 100 µm. (B) The fluorescence intensity of Rhodamine staining in HCECs. (C) OCR in siAMPK transfected HCECs versus siCtrl transfected HCECs. (D) Quantification of basal respiration, ATP production and maximal mitochondrial respiratory of HCECs in (C) . (E, F) Representative flow cytometry analysis images and quantification of MitoTracker Green staining in HCECs transfected with siCtrl and siAMPK. (G–I) Western blot and quantitative analysis of protein expression of MFN2, OPA1, DRP1, MFF, and Complex IV in HCECs with β-actin as a loading control. (J) Measurement of ATP levels in corneal epithelium of Flox and KO mice. (K–M) Western blot and quantitative analysis of protein expression of MFN2, OPA1, DRP1, MFF, and Complex IV in corneal epithelium with β-actin as a loading control. n = 6 (A–D) , n = 4 (G–I) , n = 3 (E, F, J, K–M) . Data shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Primary antibodies used in this study were as follows: p-AMPKα (2535, 1:1000), AMPKα (5831, 1:1000), phosphorylated acetyl-CoA carboxylase (p-ACC, 11818, 1:1000), ACC (3676, 1:1000), and Dynamin-related protein 1 (DRP1; 5391, 1:1000) from Cell Signaling Technology (Danvers, MA, USA); ZO-1 (21773-1-AP, 1:1000), Occludin (66378-1-lg, 1:1000), E-cadherin (CDH, 20874-1-AP, 1:1000), Mitofusion 2 (MFN2, 12186-1-AP, 1:1000), Optic atrophy 1 (OPA1, 27733-1-AP, 1:1000), MFF (17090-1-AP, 1:1000), IL-1β (16806-1-AP, 1:1000), IL-10 (60269-1-lg, 1:1000), Mitochondria Complex IV (MTCO2, 55070-1-AP, 1:1000), β-actin (HRP-60008, 1:10,000), and secondary antibodies (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG), all purchased from Proteintech (Wuhan, China).

Techniques: Staining, Fluorescence, Transfection, Flow Cytometry, Western Blot, Expressing, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: AMPK Deficiency Induces Corneal Epithelial Barrier Dysfunction by Modulating Energy Homeostasis

doi: 10.1167/iovs.67.2.47

Figure Lengend Snippet: AMPK deficiency downregulates glucose uptake and glycolysis in corneal epithelial cells. (A) Representative flow cytometry analysis images and quantification of 2-NBDG staining in HCECs transfected with siCtrl and siAMPK. (B) Activity of HK2, PFK and PKM in HCECs transfected with siCtrl and siAMPK. (C–H) Western blot and quantitative analysis of protein expression of GLUT1, HK2, PFK, PKM and PFKFB3 in corneal epithelium with β-actin as a loading control. (I) The mRNA expression of PFKFB3 in corneal epithelium of Flox and KO mice. n = 5 (A and B) , n = 3 (C–H) . n = 4 (I) . Data shown as mean± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Primary antibodies used in this study were as follows: p-AMPKα (2535, 1:1000), AMPKα (5831, 1:1000), phosphorylated acetyl-CoA carboxylase (p-ACC, 11818, 1:1000), ACC (3676, 1:1000), and Dynamin-related protein 1 (DRP1; 5391, 1:1000) from Cell Signaling Technology (Danvers, MA, USA); ZO-1 (21773-1-AP, 1:1000), Occludin (66378-1-lg, 1:1000), E-cadherin (CDH, 20874-1-AP, 1:1000), Mitofusion 2 (MFN2, 12186-1-AP, 1:1000), Optic atrophy 1 (OPA1, 27733-1-AP, 1:1000), MFF (17090-1-AP, 1:1000), IL-1β (16806-1-AP, 1:1000), IL-10 (60269-1-lg, 1:1000), Mitochondria Complex IV (MTCO2, 55070-1-AP, 1:1000), β-actin (HRP-60008, 1:10,000), and secondary antibodies (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG), all purchased from Proteintech (Wuhan, China).

Techniques: Flow Cytometry, Staining, Transfection, Activity Assay, Western Blot, Expressing, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: AMPK Deficiency Induces Corneal Epithelial Barrier Dysfunction by Modulating Energy Homeostasis

doi: 10.1167/iovs.67.2.47

Figure Lengend Snippet: AMPK deficiency induces elevated inflammation in corneal epithelial cells. (A, B) The mRNA expression of IL1β and TNFα in HCECs transfected with siCtrl and siAMPK. (C, D) The mRNA expression of IL1β and TNFα in corneal epithelium of Flox and KO mice. (E, F) Western blot and quantitative analysis of protein expression of IL1β and IL10 in HCECs with β-actin as a loading control. (G, H) Western blot and quantitative analysis of protein expression of IL1β and IL10 in corneal epithelium with β-actin as a loading control. n = 5 (A–D) , n = 4 (E and F) , n = 3 (G and H) . Data was shown as mean± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Primary antibodies used in this study were as follows: p-AMPKα (2535, 1:1000), AMPKα (5831, 1:1000), phosphorylated acetyl-CoA carboxylase (p-ACC, 11818, 1:1000), ACC (3676, 1:1000), and Dynamin-related protein 1 (DRP1; 5391, 1:1000) from Cell Signaling Technology (Danvers, MA, USA); ZO-1 (21773-1-AP, 1:1000), Occludin (66378-1-lg, 1:1000), E-cadherin (CDH, 20874-1-AP, 1:1000), Mitofusion 2 (MFN2, 12186-1-AP, 1:1000), Optic atrophy 1 (OPA1, 27733-1-AP, 1:1000), MFF (17090-1-AP, 1:1000), IL-1β (16806-1-AP, 1:1000), IL-10 (60269-1-lg, 1:1000), Mitochondria Complex IV (MTCO2, 55070-1-AP, 1:1000), β-actin (HRP-60008, 1:10,000), and secondary antibodies (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG), all purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Expression and function of Toll-like receptors in peripheral blood mononuclear cells in patients with ankylosing spondylitis

doi: 10.3892/mmr.2019.10631

Figure Lengend Snippet: TNF-α mediates TLR expression and NF-κB p65 signaling in PBMCs. (A) Western blotting was performed to detect the protein levels of TLR3, TLR4, TLR5, p65 and p-p65. Protein expression of (B) TLR3, (C) TLR4, (D) TLR5 and (E) p-p65/p65 presented as bar diagrams. β-actin was used as an internal control. *P<0.05, **P<0.01 vs. control. NF-κB, nuclear factor-κB; p-, phosphorylated; PBMC, peripheral blood mononuclear cell; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-α.

Article Snippet: The membranes were blocked in 5% dried skimmed milk in TBS buffer at 37°C for 1 h and incubated overnight at 4°C with antibodies against TLR3 (1:700; ab62566, Abcam), TLR4 (1:600; ab13556, Abcam), TLR5 (1:800; ab62460, Abcam), p65 (1:1,000; ab32536, Abcam), phosphorylated (p)-p65 (1:600; ab86299, Abcam) and β-actin (1:1,000; MAB8969, R&D Systems, Inc.).

Techniques: Expressing, Western Blot, Control

Journal: Virology

Article Title: SNAP23 is essential for germination of EV-D68 replication organelles.

doi: 10.1016/j.virol.2022.11.011

Figure Lengend Snippet: Fig. 2. SNAP23 expression is not altered by EV-D68 infection but SNAP23 knockdown inhibits VP3 expression. (A) Cells were infected with EV-D68 over a course of 5 h (MOI = 25). Samples were collected at each indicated time point for Western blot analysis. (B) Cells were transfected with non-targeting or SNAP23 siRNAs for 48 h and then infected with EV-D68 (MOI = 10) for 4 h. Cell lysates were collected at the end of the infection and used for Western blot analysis. Subsets of non-targeting and siSNAP23 transfected cells in B were also treated with 2 mM guanidine hydrochloride (GuHCl) as a control to inhibit viral replication. (C) SNAP23 bands from three indepen dent experiments, completed for (A), were semi- quantified and normalized to beta-actin. (D) Bands from full length SQSTM1, in (B), were semi- quantified from three independent experiments and normalized to beta-actin. (E) VP3 bands were semi- quantified from three independent experiments and independent from the blots, in (B), probed against SQSTM1 due to the cleavage product from SQSTM1 is close in size to VP3. n = 3 independent experiments. Unless noted by an asterisk, results were otherwise not statistically significant. * = p-value <0.05 and error bars represent the mean 土SEM.

Article Snippet: Primary antibodies used include: SNAP23 ([EPR8538], ab131242, Abcam), LC3 (NB600-1384, Novus), SQSTM1 (H00008878-M01, Abnova), Beta Actin (NB600-532, Novus), and Enterovirus pan Monoclonal Antibody to detect VP3 (MA5-18206, Invitrogen).

Techniques: Expressing, Infection, Knockdown, Western Blot, Transfection, Control