Journal: Frontiers in Endocrinology

Article Title: Transcriptome Analysis Reveal Candidate Genes and Pathways Responses to Lactate Dehydrogenase Inhibition (Oxamate) in Hyperglycemic Human Renal Proximal Epithelial Tubular Cells

doi: 10.3389/fendo.2022.785605

Figure Lengend Snippet: The protein expression levels of peroxisome proliferator-activated receptor-g coactivator 1α (PGC1α), caspase 3 (CASP3), B-cell lymphoma 2 apoptosis regulator (BCL2), BCL2 associated X apoptosis regulator (BAX), beclin1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3) in HK-2 cells treated with low D-glucose (LG) with different concentrations of oxamate (LGOXA-0mM, LGOXA-20mM, LGOXA-40mM and LGOXA-80mM) and high D-glucose (HG) with different concentrations of oxamate (HGOXA-0mM, HGOXA-20mM, HGOXA-40mM and HGOXA-80mM) for 24 hours. (A) Western blot gels. (B) Relative grey values. * indicates P -value < 0.05 after student’s t -test.

Article Snippet: The primary antibodies [Anti-PGC1α (ab54481), Anti-CASP3 (9665), Anti-BCL2 (ab59348), Anti-BAX (GB11690), Anti-BECN1 (bs-1353R), Anti-MAP1LC3 (14600-1-ap) and Anti-β-actin (GB12001)] and all secondary antibodies were purchased from abcom (Cambridge, MA, USA), Proteintech (Wuhan, China), BIOSS (Boston, Massachusetts, USA) and Servicebio (Wuhan, China), respectively.

Techniques: Expressing, Western Blot

Journal: Journal of Virology

Article Title: Swine MicroRNAs ssc-miR-221-3p and ssc-miR-222 Restrict the Cross-Species Infection of Avian Influenza Virus

doi: 10.1128/JVI.01700-20

Figure Lengend Snippet: AIV infection and ssc-miR-221-3p/222 transfection induce PAM cell apoptosis. (A and B) PAM cells were infected with AIV (H5N1, H7N9, and H9N2) as indicated at an MOI of 1 for 24 h. (C and D) PAM cells were transfected with miRNA mimics or control at 60 nM for 24 h. The cells were collected by centrifugation and were resuspended in 100 μl of 1× binding buffer supplemented with 5 μl of PE annexin V and 5 μl 7-AAD. Fluorescence of the stained cells was then analyzed using flow cytometry. The total protein was collected and Bcl2, caspase 3, and cleave caspase 3 protein levels were determined by Western blotting, with β-actin as an internal control. Data are presented as means ± SD of results from three independent experiments. Statistical significance was based on two-way ANOVA (**, P < 0.01; ***, P < 0.001).

Article Snippet: Primary antibodies included anti-β-actin (1:1,000; AA128; Beyotime), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:1,000; AF0006; Beyotime), anti-NP (WB, 1:1,000; A01506; GenScript), anti-NF-κB P65 (WB, 1:1,000; ChIP, 1:100; 6956; Cell Signaling Technology), anti-phospho-NF-κB P65 (Ser536) (1:1,000; 3033; Cell Signaling Technology), anti-IκBα (1:1,000; 4814; Cell Signaling Technology), anti-phospho-IκBα (Ser32) (1:1,000; bsm-52169R; Bioss), anti-HMBOX1 (1:1,000; bs-18047R; Bioss), anti-Bcl2 (1:1,000; bsm-33047M; Bioss), anti-caspase 3/P17/P19 (1:1,000; 19677-1-AP; Proteintech), and anti-NP (IFA, 1:5,000; ab20343; Abcam).

Techniques: Infection, Transfection, Centrifugation, Binding Assay, Fluorescence, Staining, Flow Cytometry, Western Blot

Journal: Journal of Virology

Article Title: Swine MicroRNAs ssc-miR-221-3p and ssc-miR-222 Restrict the Cross-Species Infection of Avian Influenza Virus

doi: 10.1128/JVI.01700-20

Figure Lengend Snippet: ssc-miR-221-3p and ssc-miR-222 target HMBOX1. (A) Selection of host targets of ssc-miR-221-3p/222. HEK293T cells were cotransfected with luciferase reporter vectors pGL3 containing the seven potential target genes, pRL-TK, and 80 nM miRNA mimics or 80 nM randomized oligonucleotide as a mimic control. Cell lysates were harvested for dual-luciferase assays at 24 hpi. (B) Alignments of HMBOX1 binding sites with miR-221-3p/222. Perfect matches are indicated by a line between the 3′ UTR of HMBOX1 (HMBOX1-wt) and miR-221-3p/222. Mutations (in red in HMBOX1-mut) were made in the seed region of the miR-221-3p/222 binding site for the reporter assay. 293T cells were cotransfected with 80 nM ssc-miR-221-3p/222 mimic or randomized oligonucleotide, the reporter plasmid HMBOX1 or HMBOX1-mut, and pRL-TK. Cell lysates were harvested for dual-luciferase assays at 24 hpi. (C) Conserved miR-221-3p/222 binding sites in the 3′ UTR of HMBOX1. (D) ssc-miR-221-3p/222 inhibited HMBOX1 expression in PAM cells. PAM cells were transfected with miRNA controls or miR-221-3p/222 mimics at 80 nM. At 24 h after transfection, cells were harvested, and the cytosolic proteins were examined by Western blotting using anti-HMBOX1 polyclonal antibodies. GAPDH expression was used as an internal control. (E) PAM cells were transfected with 60 nM HMBOX1-specific siRNA (siHMBOX1) or scrambled siRNA (siCtrl) as a control for 24 h, followed by H5N1 AIV infection (MOI, 1) for 24 h. PAM cells were harvested for Western blotting against HMBOX1, NP, Bcl2, caspase 3, and cleave caspase 3, with β-actin as an internal control. Data are presented as means ± SD of results from three independent experiments. Statistical significance was based on two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Primary antibodies included anti-β-actin (1:1,000; AA128; Beyotime), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:1,000; AF0006; Beyotime), anti-NP (WB, 1:1,000; A01506; GenScript), anti-NF-κB P65 (WB, 1:1,000; ChIP, 1:100; 6956; Cell Signaling Technology), anti-phospho-NF-κB P65 (Ser536) (1:1,000; 3033; Cell Signaling Technology), anti-IκBα (1:1,000; 4814; Cell Signaling Technology), anti-phospho-IκBα (Ser32) (1:1,000; bsm-52169R; Bioss), anti-HMBOX1 (1:1,000; bs-18047R; Bioss), anti-Bcl2 (1:1,000; bsm-33047M; Bioss), anti-caspase 3/P17/P19 (1:1,000; 19677-1-AP; Proteintech), and anti-NP (IFA, 1:5,000; ab20343; Abcam).

Techniques: Selection, Luciferase, Binding Assay, Reporter Assay, Plasmid Preparation, Expressing, Transfection, Western Blot, Infection