Journal: Communications Biology

Article Title: SERPINA3-ANKRD11-HDAC3 pathway induced aromatase inhibitor resistance in breast cancer can be reversed by HDAC3 inhibition

doi: 10.1038/s42003-023-05065-w

Figure Lengend Snippet: a The expression of SERPINA3 is positively correlated with ESR1 in breast cancer (retrieved from cBioPortal). b SERPINA3 exhibits significant higher expression in ER+ breast cancer ( n = 7247) than ER− subtype ( n = 2710) (retrieved from bc-GenExMiner v4.9). c QPCR and d Western blot results revealed a decrease of ERα expression in LTED cells, when compared with their parental cells. e The expression of ESR1 could be effectively inhibited by the siRNAs in MCF-7 and T47D cells. f QPCR and g western blot results showed that siRNA knockdown of ESR1 in MCF-7 and T47D effectively downregulated SERPINA3 expression. GAPDH and SERPINA3 were blotted from different gels for their close molecular weight. h SERPINA3 mRNA expression decreased with extended estrogen deprivation incubation in MCF-7 and T47D. i The details of the predicted ERα binding sequence in SERPINA3 promoter obtained from JASPAR. j Comparison of ERα binding motif and the predicted ERα binding sequence in SERPINA3 promoter. k Dual luciferase reporter assay verified the physical interaction of ERα and SERPINA3 promoter at −462 to −446 bp. The promoter plasmid or its mutant type were co-transfected with pCDH empty vector or ESR1-expressing plasmid as indicated. Luciferase assays were then performed. pCDH-Vector: pCDH-CMV-MCS-EF1-puro; pCDH-ESR1: pCDH-CMV-MCS-EF1-puro-ESR1. Data are representative of n = 3 biologically independent experiments and presented as mean ± SD; statistical significance is determined by unpaired Student’s t test ( p < 0.05 *, p < 0.01 **, p < 0.001 ***).

Article Snippet: ERα over-expressing plasmids pCDH-CMV-MCS-EF1-PURO-ESR1 and its empty vector pCDH were purchased from OriGene (Rockville, MD).

Techniques: Expressing, Western Blot, Knockdown, Molecular Weight, Incubation, Binding Assay, Sequencing, Comparison, Luciferase, Reporter Assay, Plasmid Preparation, Mutagenesis, Transfection

Journal: The Journal of antimicrobial chemotherapy

Article Title: Antiviral activity of baicalin against influenza virus H1N1-pdm09 is due to modulation of NS1-mediated cellular innate immune responses.

doi: 10.1093/jac/dkt534

Figure Lengend Snippet: Figure 4. Effect of baicalin in vitro and in vivo on NS1-induced Akt phosphorylation. (a) A549 cells infected with the H1N1-pdm09 strain at 1.0 moi were incubated with or without baicalin (10 mM). Cell lysates prepared at specific times post-infection were subjected to western blotting using phospho-Akt (Ser 473), NP and Akt antibodies. (b) Effect of baicalin on the PI3K/Akt pathway during influenza virus infection was evaluated in BALB/c mice by treating the mice with baicalin at 40 mg/kg/day and, after 24 h, subjecting lung homogenates to western blotting using phospho-Akt (Ser 473), M1 and Akt antibodies. Band intensity was quantified using Quantity One software. (c) A549 cells transfected with pcD-NS1 or vector (pcDNA6) control were incubated with or without baicalin (0.5–16 mM). Cell lysates prepared after 24 h were subjected to western blotting using phospho-Akt (Ser 473), NS1 and Akt antibodies. (d) The serum-starved A549 cells infected with H1N1-pdm09 (1.0 moi) were treated with baicalin (10 mM) or DMSO vehicle for 24 h. The NS1 was immunoprecipitated using NS1 antibody and immunoblotted with p-85b antibody to confirm association. (e) A549 cells transfected with pcD-NS1 or empty vector (pcDNA6) control were treated with baicalin (10 mM) or DMSO (control) for 24 h. Cell lysates were subjected to immunoprecipitation with NS1 antibody and immunoblotted with p-85b antibody. The membrane was reprobed with anti-mouse secondary antibody as a loading control for immunoprecipitation. The blot shown is representative of three independent experiments. P,0.05 was considered statistically significant.

Article Snippet: Similarly, PKR expression was low, which could be due to both increased p-STAT3 activity, which suppresses autophagy through PKR, as well as reduced viral replication.76 In addition to the IRF-3/RNase L pathway, NS1 also modulates IFN induction through activation of the SOCS-3 protein,77,78 which is consistent with our results in which reduced SOCS-3 expression 450 000.00(a) (b) (c) Normal NS1 protein Q40H NS1 20 mM BA + Q40H NS1 10 mM BA + NS1 20 mM BA + NS1 400 000.00 350 000.00 300 000.00 250 000.00 200 000.00 150 000.00 100 000.00 50 000.00 0.00 450 000.00 400 000.00 350 000.00 300 000.00 250 000.00 200 000.00 150 000.00 100 000.00 50 000.00 0.00 30 0 30 5 31 0 31 5 32 0 32 5 33 0 33 5 Wavelength (nm) Fl uo re sc en ce in te ns ity Fl uo re sc en ce in te ns ity 34 0 34 5 35 0 35 5 36 0 36 5 37 0 37 5 38 0 38 5 39 0 39 5 40 0 30 0 30 5 31 0 31 5 32 0 32 5 33 0 33 5 Wavelength (nm) Baicalin 10 mM pcDNA (empty vector) pcD-NS1 (full length) pcD-R46G-NS1 pcD-Q40H-NS1 pcD-S42P-NS1 p-85b IgG NS1 (input lysate) + + – – – – – – + – – – + – – + – – + – – – + – + – – – – + 34 0 34 5 35 0 35 5 36 0 36 5 37 0 37 5 38 0 38 5 39 0 39 5 40 0 Normal NS1 protein S42P NS1 20 mM BA + S42P NS1 10 mM BA + NS1 20 mM BA + NS1 Figure 7.

Techniques: In Vitro, In Vivo, Phospho-proteomics, Infection, Incubation, Western Blot, Virus, Software, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Membrane

Journal: The Journal of antimicrobial chemotherapy

Article Title: Antiviral activity of baicalin against influenza virus H1N1-pdm09 is due to modulation of NS1-mediated cellular innate immune responses.

doi: 10.1093/jac/dkt534

Figure Lengend Snippet: Figure 7. Glutamine (at position 40) and serine (at position 42) interact with NS1. Analysis of the change in fluorescence spectroscopy spectra, by excitation at a wavelength of 290 nm and emission at a wavelength of 300–400 nm, of NS1 protein (a and b), Q40H NS1 mutant (a) and S42P NS1 mutant (b) in the presence or absence of baicalin (BA), revealed that changes in amino acids at positions 40 and 42 disrupted the binding of NS1 and baicalin. (c) A549 cells transfected with pcD-R46GNS1, pcD-Q40H-NS1, pcD-S42P-NS1 or empty vector (pcDNA6) and treated with baicalin (10 mM) for 24 h. For a positive control, A549 cells transfected with full-length pcD-NS1 in the absence of baicalin were used. Cell lysates were subjected to immunoprecipitation with NS1 antibody and immunoblotted with p-85b antibody. Cell lysates were immunoblotted with NS1 to confirm expression of NS1 in transfected cells. Results are representative of three independent experiments.

Article Snippet: Similarly, PKR expression was low, which could be due to both increased p-STAT3 activity, which suppresses autophagy through PKR, as well as reduced viral replication.76 In addition to the IRF-3/RNase L pathway, NS1 also modulates IFN induction through activation of the SOCS-3 protein,77,78 which is consistent with our results in which reduced SOCS-3 expression 450 000.00(a) (b) (c) Normal NS1 protein Q40H NS1 20 mM BA + Q40H NS1 10 mM BA + NS1 20 mM BA + NS1 400 000.00 350 000.00 300 000.00 250 000.00 200 000.00 150 000.00 100 000.00 50 000.00 0.00 450 000.00 400 000.00 350 000.00 300 000.00 250 000.00 200 000.00 150 000.00 100 000.00 50 000.00 0.00 30 0 30 5 31 0 31 5 32 0 32 5 33 0 33 5 Wavelength (nm) Fl uo re sc en ce in te ns ity Fl uo re sc en ce in te ns ity 34 0 34 5 35 0 35 5 36 0 36 5 37 0 37 5 38 0 38 5 39 0 39 5 40 0 30 0 30 5 31 0 31 5 32 0 32 5 33 0 33 5 Wavelength (nm) Baicalin 10 mM pcDNA (empty vector) pcD-NS1 (full length) pcD-R46G-NS1 pcD-Q40H-NS1 pcD-S42P-NS1 p-85b IgG NS1 (input lysate) + + – – – – – – + – – – + – – + – – + – – – + – + – – – – + 34 0 34 5 35 0 35 5 36 0 36 5 37 0 37 5 38 0 38 5 39 0 39 5 40 0 Normal NS1 protein S42P NS1 20 mM BA + S42P NS1 10 mM BA + NS1 20 mM BA + NS1 Figure 7.

Techniques: Spectroscopy, Mutagenesis, Binding Assay, Transfection, Plasmid Preparation, Positive Control, Immunoprecipitation, Expressing