Journal: bioRxiv

Article Title: ProPER: Programmable, multiplexed detection of molecular proximities and RNA life-cycle stages in situ

doi: 10.64898/2026.04.24.718482

Figure Lengend Snippet: (a) Principle of proximity detection by in situ PER (left) and reaction mechanism (right). The docking sites for primer and hairpin need to be in close physical proximity for the catalytic primer extension to take place. Extended DNA concatemer is detected by fluorescently labeled imager oligos. When a cognate primer and hairpin pair are in close proximity, PER – takes place. The polymerase extends the primer with copies of sequence a and generates a linear ssDNA concatemer through autonomous and isothermal cycles of: (i) primer-hairpin binding, (ii) extension of the primer by a strand-displacing polymerase (in this work BST large fragment) using the hairpin’s a* sequence as a template, (iii) branch migration, (iv) dissociation of the primer from the hairpin. (b) Applications for proximity detection by ProPER. Primer and hairpin are docked to RNA by in situ hybridization of DNA probes or to proteins via DNA-labeled antibodies to detect proximity of the molecules or coincidence of probes on the same target. Bottom: In detection cascades different sequences are attached in a programmed order. Right: The life cycle of an RNA can be revealed by multiplexed detection of proximity to life-cycle markers. (c) In situ RNA FISH and ProPER for CBX5 gene in HeLa cells under limiting probe numbers. SABER-FISH was performed with in vitro pre-extended concatemers, whereas for ProPER primers were extended in situ after docking to a single site (ProPER medium) or multiple sites on a pre-extended primary probe (ProPER high). Representative images show maximum projections of FISH/ProPER signal (orange to white gradient) and DAPI (cyan). (d) Negative controls for the ProPER medium condition, where primer, hairpin dock or polymerase were left out. I Raw signal intensity of the spots (in arbitrary fluorescence units, a.u.) and (f) spot counts per cell showed higher values for ProPER compared to SABER-FISH with a small number of FISH probes (5-6 oligo probes). Signal to background ratio (S/B) is noted above each curve (see Methods ). Mann-Whitney-Wilcoxon test, two-sided, * p<0.05, *** p<0.001 (exact p-values in Supplementary Table S3 ). N=105-782 cells per condition. Scale bars 10 μm.

Article Snippet: Final concentrations in 200 μL reaction volume were 1× PBS, 480U/ml Bst LF polymerase (NEB, M0275M), 10 mM MgSO4, 0.6 mM dNTPs (dATP, dCTP, dTTP), 0.1 μM Clean.G hairpin (IDT), 0.6-1.0 μM hairpin and 0.75 μM primer.

Techniques: In Situ, Labeling, Sequencing, Binding Assay, Migration, RNA In Situ Hybridization, In Vitro, Fluorescence, MANN-WHITNEY

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 1. TNF or LPS activates IKK. (A) DPSCs were treated with TNF (10 ng/mL) and P. gingivalis LPS (1 g/mL) for the indicated times. Fifty-g aliquots of protein extracts were probed with polyclonal antibodies against IB (1:1000) or monoclonal antibodies against phospho-specific antibodies (1:1000). For loading control, the blots were stripped and re-examined with monoclonal antibodies against - tubulin (1:15, 000). (B) E. coli LPS activates IKK. DPSCs were treated with E. coli LPS (200 ng/mL) or TNF for the indicated time points. Western blot analysis was performed as described in (A).

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Bioprocessing, Control, Western Blot

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 2. TNF or LPS induces the nuclear translocation of NF-B. (A) DPSCs were treated with TNF (10 ng/mL) for the indicated time. Five- g aliquots of nuclear proteins were incubated with 32P-labeled NF-B oligonucleotide probes. For the supershift assay, nuclear proteins were pre-incubated with polyclonal antibodies against p65 for 10 min and then probed with32P-labeled NF-B oligonucleotide probes. (B) DPSCs were treated with P. gingivalis LPS (1 g/mL) for the indicated times. EMSA was performed as described in (A).

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Translocation Assay, Incubation, Labeling

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 3. LPS induces IL-8 expression in DPSCs. (A) DPSCs were treated with TNF or P. gingivalis LPS for the indicated times. Fifty-g aliquots of protein extracts were probed with polyclonal antibodies against phospho-specific p65. For loading control, the membrane was stripped and re-probed with polyclonal antibodies against p65. (B) DPSCs were treated with TNF for the indicated times. Ten-g aliquots of total RNAs were hybridized with 32P-labeled IL-8 cDNA probes. For loading control, the blot was stripped and re-probed with 32P-labeled glyceraldehyde-3- phosphate dehydrogenase (GAPDH) cDNA probes. (C) DPSCs were treated with P. gingivalis LPS for the indicated times. Northern blot analysis was performed as described in (B). (D) DPSCs were treated with TNF for the indicated times. Total RNAs were probed with 32P-labeled IL- 6 cDNA probes. (E) DPSCs were treated with E. coli LPS for the indicated times. Total RNAs were probed with 32P-labeded IL-6 cDNA probes.

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Expressing, Control, Membrane, Labeling, Northern Blot

Journal: Nature Communications

Article Title: An antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo

doi: 10.1038/s41467-024-46533-z

Figure Lengend Snippet: a Schematic illustration of the protocol of PBS, AFMFlip pCas9-gHBV , MFlip-a pCas9-gHBV , and MFlip-b pCas9-gHBV treating HBV infection. b Sanger DNA sequencing results of the gene editing at HBV-targeting sites amplified from the liver tissue DNA of mice treated with PBS and AFMFlip pCas9-gHBV . The experiment was repeated three times independently with similar results. c Tracking of indels by decomposition (TIDE) analysis of sequencing results at the targeted HBV genome from liver tissue DNA of mice treated with AFMFlip pCas9-gHBV . The editing efficiency for TIDE analysis was calculated online ( https://tide.nki.nl/ ). The experiment was repeated three times independently with similar results. d T7 endonuclease I (T7E1) assay at the HBV-targeting site in the liver tissue DNA from mice treated with PBS and AFMFlip pCas9-gHBV . The cutting efficiency of indel was determined by band densitometry using ImageJ software. The experiment was repeated three times independently with similar results. e – g Analysis of HBV-related viral loads in the serum from HBV-replication mice treated with PBS, AFMFlip pCas9-gHBV , MFlip-a pCas9-gHBV , and MFlip-b pCas9-gHBV , including HBsAg ( e ), HBeAg ( f ), and HBV DNA ( g ). Data are presented as mean ± SD and statistically analyzed using one-way ANOVA ( n = 5 biologically independent animals). h Immunofluorescence staining of HBsAg in the liver sections harvested from HBV-replication mice treated with PBS, AFMFlip pCas9-gHBV , MFlip-a pCas9-gHBV , and MFlip-b pCas9-gHBV . The experiment was repeated three times independently with similar results. i – k Determination analysis of HBsAg ( i ), HBeAg ( j ), HBV DNA ( k ) from liver tissues. All values shown were normalized to the negative control group with PBS treatment. Data are presented as mean ± SD and statistically analyzed using one-way ANOVA ( n = 5 biologically independent animals). Source Data are provided as a Source Data file.

Article Snippet: DNA fragment with CRISPR/Cas9 target location was PCR amplified using target-specific primers and purified by Tiangen gel extraction kit (Tiangen).

Techniques: Infection, DNA Sequencing, Amplification, Sequencing, Software, Immunofluorescence, Staining, Negative Control