Journal: bioRxiv

Article Title: HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

doi: 10.1101/2025.07.16.665136

Figure Lengend Snippet: A . Chemical structure of C19, generated using ChemDraw software. B.C. Docking poses of C19 targeting the ZO-1 PDZ2 swapped dimer. Panels show the best predicted binding poses of C19 when docked to the ZO-1 PDZ2 dimer either in its free form ( B ) or in complex with the E protein PBM ( C ), using DOCK6 simulations. The PDZ domains are shown in cartoon representation. The compound is positioned within key binding regions of the ZO-1 PDZ2 dimer, preferentially occupying the PBM binding site. D . HTRF inhibition dose-response curve of C19. The assay was performed in 96-well, white, low-volume HTRF plates with 5 µL of purified GST-ZO-1-PDZ2 (final concentration: 3 nM), 5 µL of purified His₆-Ecyto (final concentration: 60 nM), and 1 µL of C19, tested in a 10-point, twofold serial dilution (100 µM to 195 nM). The resulting curve represents the percentage of residual interaction (%R) based on the normalized FRET signal. Sigmoidal curve was generated using nonlinear regression in Prism software, plotting C19 concentration against the normalized response (n=1). E.F . Antiviral activity of C19. The assay was conducted in 96-well culture plates using Vero-E6 cells (40,000 cells/well) infected with a NanoLuciferase-expressing recombinant SARS-CoV-2 virus at a MOI of 0.001. Cells were incubated for 72 hours in the presence of C19, applied as a 9-point, twofold serial dilution ranging from 20 µM to 78 nM. Viral replication was assessed by measuring luminescence in the cell culture supernatant using the Nano-Glo assay kit ( E ). Data were normalized to both infected untreated and uninfected untreated control conditions. Sigmoidal curve was generated using nonlinear regression in Prism software, plotting C19 concentration against the normalized luminescence signal (n=3). Viral quantification was performed using plaque assay on Vero-E6 cells (F). Plaques were observed at 3 days post-infection (dpi) following infection with serial dilutions of supernatants collected from infected non-treated (INT) or infected treated (IT) cells, treated for 72 hours with 20 µM of compound C19. Viral titers are reported as plaque-forming units per milliliter (PFU/mL). Horizontal lines represent the mean ± SEM, and dashed lines indicate the limit of detection (n=3; nd = not detected). Statistical significance was determined by t-test (p < 0.005).

Article Snippet: Cells were cultured for 72 hours post-infection, after which luminescence was measured following the manufacturer’s protocol (N1120, Promega).

Techniques: Generated, Software, Binding Assay, Inhibition, Purification, Concentration Assay, Serial Dilution, Activity Assay, Infection, Expressing, Recombinant, Virus, Incubation, Cell Culture, Glo Assay, Control, Plaque Assay

Journal: Scientific Reports

Article Title: LncRNA CTD-2555A7.2 promotes bone formation with LncRNA-specific cascade amplification strategy

doi: 10.1038/s41598-025-05826-z

Figure Lengend Snippet: CTD-2555A7.2 bind with multiple miR-381-3p and promoted bone formation. A Sequence structure of CTD-2555A7.2. B miR-381-3p expression levels of hMSCs treated with CTD-2555A7.2 over-expression plasmid (compared with pCDNA3.1 plasmid), as detected by RT-PCR (mean ± SD, n = 3). vec: blank plasmid. CTD: plasmid containing CTD-2555A7.2 fell length sequence. C miR-381-3p expression levels of hMSCs treated with CTD-2555A7.2 siRNA (compared with negative control siRNA, mean ± SD, n = 3). D miR-381-3p expression levels of hMSCs treated with CTD-2555A7.2 full length, CTD-2555A7.2 non-binding region, CTD-2555A7.2 binding region and CTD-2555A7.2 mutant binding region over-expression plasmids respectively (compared with pCDNA3.1 plasmid), as detected by RT-PCR (mean ± SD, n = 3). non: plasmid containing CTD-2555A7.2 non-binding region. bind: plasmid containing CTD-2555A7.2 binding region. bind mut: plasmid containing CTD-2555A7.2 mutant binding region. E Binding effect of miR-381-3p and CTD-2555A7.2 binding region, as detected by luciferase reporter assay (mean ± SD, n = 3). Luc-mut: pMIR-Report luciferase reporter plasmid containing one mutant miR-381-3p binding repeat sequence of CTD-2555A7.2. Luc-WT: luciferase reporter plasmid containing one wild-type miR-381-3p binding repeat sequence of CTD-2555A7.2. Ago-NC: cells treated with agomiR-NC. Ago-381-3p: cells treated with agomiR-381-3p. F,G Binding effect of miR-381-3p and CTD-2555A7.2 different regions (compared with pMIR-Report luciferase reporter plasmid) and treated by agomiR-381-3p (left) or antagomiR-381-3p (right), as detected by luciferase reporter assay (mean ± SD, n = 3). Atgo-NC: cells treated with antagomiR-NC. Atgo-381-3p: cells treated with antagomiR-381-3p. H ALP and Alizarin Red staining of hMSC cell treated with CTD-2555A7.2 exprssion plasmids of different regions, as detected by ALP staining and Alizarin Red staining. I. TCF7 activities of hMSCs treated with CTD-2555A7.2 exprssion plasmids of different regions, as detected by luciferase reporter assay (mean ± S.D., n = 8). J,K Calvarial bone mineral apposition rates of C57BL/6 mice treated with CTD-2555A7.2 exprssion plasmids of different regions (mean ± SD, n = 4). Scale bar: 10μm.* P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Luciferase reporter assay in vitro (with hMSC) and in vivo (with isolated BMSC) were performed as previously (E1910 and N1120, Promega, Fitchburg, WI, USA) .

Techniques: Sequencing, Expressing, Over Expression, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Staining

Journal: Scientific Reports

Article Title: LncRNA CTD-2555A7.2 promotes bone formation with LncRNA-specific cascade amplification strategy

doi: 10.1038/s41598-025-05826-z

Figure Lengend Snippet: miR-381-3p inhibited four target genes of Wnt signaling pathway. A, C Protein expression of APC, LEF1, LRP6 and wnt5a of hMSCs treated with mimic-381-3p (compared with mimic-NC), as detected by western blot (mean ± S.D., n = 3). B, D Protein expression of APC, LEF1, LRP6 and wnt5a of hMSCs treated with inhibitor-381-3p, as detected by western blot(mean± S.D., n > 3). E-H Binding effect of miR-381-3p and 3′UTR sequences of Apc , Lef1 , Lrp6 and wnt5a , as detected by luciferase reporter assay (mean ± S.D., n = 3). Luc-vec: empty luciferase reporter plasmid. Luc-mut: luciferase reporter plasmid containing mutant 3′UTR. Luc-WT: luciferase reporter plasmid containing wild-type 3′UTR.* P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Luciferase reporter assay in vitro (with hMSC) and in vivo (with isolated BMSC) were performed as previously (E1910 and N1120, Promega, Fitchburg, WI, USA) .

Techniques: Expressing, Western Blot, Binding Assay, Luciferase, Reporter Assay, Plasmid Preparation, Mutagenesis

Journal: Scientific Reports

Article Title: LncRNA CTD-2555A7.2 promotes bone formation with LncRNA-specific cascade amplification strategy

doi: 10.1038/s41598-025-05826-z

Figure Lengend Snippet: nCAR/anti381 and CTD-2555A7.2 binding sequence rescued osteoporosis. A ALP and Alizarin Red staining (left) of MC3T3-E1 cells treated with recombinant miR-381-3p inhibitor (compared with MSA), as detected by ALP staining and Alizarin Red staining, and Alp / Runx2 expression levels of MC3T3-E1 cells treated with recombinant miR-381-3p inhibitor, as detected by RT-PCR (right, mean ± S.D., n = 3). MSA: tRNAMet fused Sephadex aptamer. nCAR/anti381: novel recombinant miR-381-3p inhibitor. B ALP and Alizarin Red staining (left), and Alp / Runx2 expression levels (right) of hMSCs treated with recombinant miR-381-3p inhibitor (mean ± S.D., n = 3). C,D Femoral trabecular mineral apposition rate of C57BL/6 mice after OVX and recombinant miR-381-3p inhibitor treatment (mean ± S.D., n = 4). BL (Baseline): sacrifice before RNA treatment. Sham: sham OVX operation group. OVX: OVX group. Mock: transfection reagent control group. MSA: empty recombinant tRNA treated group. nCAR/anti381: novel recombinant miR-381-3p inhibitor treated group. Scale bar: 10μm. E,F Femoral trabecular mineral apposition rate of C57BL/6 mice after OVX and CTD-2555A7.2 binding sequence and recombinant miR-381-3p inhibitor treatment (mean ± S.D., n = 4). bind: CTD-2555A7.2 binding sequence treated group. 381: novel recombinant miR-381-3p inhibitor treated group. CTD+381: CTD-2555A7.2 binding sequence and recombinant miR-381-3p inhibitor combined treated group. Scale bar: 10μm. G Expression levels of miR-381-3p in primary BMSCs of OVX mice treated with CTD-2555A7.2 binding sequence and recombinant miR-381-3p inhibitor, as detected by RT-PCR (mean ± S.D., n = 3). H TCF7 activities of primary BMSCs of OVX mice treated with CTD-2555A7.2 binding sequence and recombinant miR-381-3p inhibitor, as detected by luciferase reporter assay (mean ± S.D., n = 3).* P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Luciferase reporter assay in vitro (with hMSC) and in vivo (with isolated BMSC) were performed as previously (E1910 and N1120, Promega, Fitchburg, WI, USA) .

Techniques: Binding Assay, Sequencing, Staining, Recombinant, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Luciferase, Reporter Assay