Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Cell death induction by 1H13-survivin/BIRC5-derived peptides targeted to the cytoplasm, mitochondria, or nucleus (A) The sequences added to the 1H13-BIRC5 peptide to target it to the: (1) cytoplasm, (2) mitochondria, or (3) nucleus. The underlined sequence represents the 1H13 peptide; in red, amino acids indicate D-amino acid substitutions. (B) A549 cells were incubated for 90 min with 5 μM FITC-labeled, nucleus-targeted peptide IH13-Nuc, and were also IF-stained with anti--SMAC, anti-IP3R or anti-GM130 antibodies, and stained with DAPI to visualize the mitochondria, ER, Golgi, and nucleus, respectively. Confocal microscope images are shown, with white arrows indicating peptide co-localization with the mitochondria (SMAC). Orange and yellow arrows indicate peptide presence in the nucleus and cytosol, respectively. (C) A549 cells were incubated with the mitochondria- or nucleus-targeted 1H13-BIRC5-derived peptide for 24 h in serum-free medium, followed by a cell proliferation assay using the SRB method. (D and E) Apoptotic cell death as induced in A549 cells following incubation for 24 h with the nucleus-targeted peptide (2/3D-1H13-Nuc) in the presence or absence of the indicated concentrations of the peptides in serum-free medium and subjected to FITC–annexin V/PI staining, followed by a flow cytometry analysis. Representative histograms for control and selected peptide concentration (D) and analysis of early and late apoptotic stages are shown (E). (F and G) Cell death as induced by 2/3D-1H13-Nuc in different cell lines, A549, SH-SY5Y, U-87MG, PC-3, and HUV-EC-C (F) or Jurkat, K562, and KMH2-LC (G) were treated with the indicated concentrations of the peptide for 24 h, then subjected to cell death analysis using propidium iodide (PI) staining and flow cytometry. (H and I) A549 cells were seeded at a density of 2 × 10 5 cells per well in a 12-well plate. After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions. Twenty-four hours post-transfection, the cells were re-seeded at 1 × 10 5 cells per well in a 12-well plate. After another 24 h, the culture medium was replaced with serum-free medium, and the cells were treated with the indicated concentrations of the 2/3D-1H13-Nuc peptide. Survivin overexpression levels were assessed by immunoblotting (H), and cell death was analyzed by propidium iodide (PI) staining followed by FACS analysis (I). Results represent the means ± SEM ( n = 3).

Article Snippet: After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions.

Techniques: Derivative Assay, Sequencing, Incubation, Labeling, Staining, Microscopy, Proliferation Assay, Flow Cytometry, Control, Concentration Assay, Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: RT-qPCR and WB of SIRT3 in scramble (SCR) and si SIRT3 conditions (A). Representative images of SCR and si SIRT3 in hNE cell cultures (B). SIRT3 silencing effects on SIRT family mRNA abundance in hNECs (C). RT-qPCR and WB of empty vector (EV) and SIRT3 overexpression (OE) conditions (D). Representative images of EV and SIRT3 OE in hNE cell cultures (E). SIRT3 OE effects on SIRT family mRNA abundance in hNECs (F). Data are presented as mean±SEM . *P<0.05 vs. control group (a.u: arbitrary units).

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Quantitative RT-PCR, Plasmid Preparation, Over Expression, Control

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Overlap between down- and up-regulated DEGs (A) and DPPs (B) from SIRT3 silencing and overexpression conditions. Functional analysis of DEGs, DEPs and DPPs from SIRT3 silencing (si) (C) and overexpression (OE) (D).

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Over Expression, Functional Assay

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Functional mapping of deregulated genes (DEGs), proteins (DEPs) and phosphoproteins (DPPs) upon SIRT3 silencing conditions in hNECs.

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Functional mapping of deregulated genes (DEGs), proteins (DEPs) and phosphoproteins (DPPs) upon SIRT3 overexpressing conditions in hNECs.

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Steady state protein levels of SIRT 1-3 were measured by Western blotting (WB) at 8-24-48 hours post-treatment (A). * p<0.05 respect to untreated-hNEC control cells. Overlap between down- and up-regulated DPPs from SIRT3 silencing and overexpression upon Aβ oligomer protein stimulation in hNECs (B). Selected differentially phosphorylated proteins affected by SIRT3 modulation in Aβ-treated cells (C). Functional analysis of DPPs from SIRT3 silencing (si) and overexpression (OE) (D).

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Western Blot, Control, Over Expression, Functional Assay

Journal: bioRxiv

Article Title: Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

doi: 10.64898/2026.03.13.711520

Figure Lengend Snippet: Predictive activation profile of pathways, biofunctions and upstream regulators at the level of SIRT3 silencing and/or overexpression upon Aβ oligomer considering APP as main hub. Positive z-scores indicate potential activated pathways, whereas negative z-scores refer to predicted inhibited pathways. Based on SIRT3 silencing and overexpression datasets (A). Activation prediction of significantly altered pathways and neuronal functions (B). Systems Biology analysis were performed through the Ingenuity Pathway Analysis software .

Article Snippet: Briefly, 1 μg of either SIRT3 (HG13033-UT, Sino Biological) or control GFP (CV026, Sino Biological) plasmid DNA was mixed with Lipofectamine 3000 (Fisher, Waltham, MA, USA, L3000015) at a 1:2 ratio in DMEM and incubated for 15 min at room temperature.

Techniques: Activation Assay, Over Expression, Software

Journal: bioRxiv

Article Title: Matrix metalloproteinases proteolyze RAB proteins and contribute to cisplatin-induced ototoxicity

doi: 10.64898/2026.02.28.708770

Figure Lengend Snippet: Gelatin zymography (A, C, E) and corresponding quantitative analysis (B, D, F, G) of MMP-2 and MMP-9 activities in the conditioned media of HEI-OC1 cells following 6 h (A, B) and 48 h (C, D) of exposure to 20 μM cisplatin. Cytosolic MMP-2 and MMP-9 activities were measured following 4 h and 6 h of treatment with 20 μM cisplatin (E-G). Standard (Std) derives from conditioned medium from HT-1080 cells. H) mRNA expression of Ifit-1 was measured using RT-qPCR following a 24h exposure to 20 μM cisplatin in the presence or absence of ARP-100 (5 μM), compared to the untreated control (nil), and normalized to B2M expression. The data are represented as the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05 and ** p <0.01).

Article Snippet: For overexpression studies, the pCMV3-C-FLAG vector encoding human MMP-2 ORF (HG10082-CF, Sino Biological) was transfected into HEI-OC1 using jetPRIME Reagent (Polyplus) according to the manufacturer’s protocol.

Techniques: Zymography, Expressing, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Matrix metalloproteinases proteolyze RAB proteins and contribute to cisplatin-induced ototoxicity

doi: 10.64898/2026.02.28.708770

Figure Lengend Snippet: mRNA expression of N-terminal truncated (NTT)- Mmp-2 (A), full-length (FL)- Mmp-2 (B), and Mmp-9 (C) was measured using RT-qPCR following exposure to 20 μM cisplatin for various timepoints, compared to the untreated control (0), and normalized to B2M expression. D) Immunofluorescence staining of HEI-OC1 cells treated with 20 μM cisplatin for 6 h or untreated cells. Staining was performed using an antibody specific to MMP-2, followed by detection with a secondary antibody conjugated to Alexa Fluor 647 (red). The zoomed-in boxes indicate nuclei with an increased red signal (magenta). The data are represented as the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05).

Article Snippet: For overexpression studies, the pCMV3-C-FLAG vector encoding human MMP-2 ORF (HG10082-CF, Sino Biological) was transfected into HEI-OC1 using jetPRIME Reagent (Polyplus) according to the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Matrix metalloproteinases proteolyze RAB proteins and contribute to cisplatin-induced ototoxicity

doi: 10.64898/2026.02.28.708770

Figure Lengend Snippet: (A) IL-6 secretion in cells transfected with either an empty vector (EV) or an Mmp-2 -expressing vector and treated with 100 μM cisplatin or left untreated (nil) for 24 h. (B, C) IL-6 secretion in cells pre-treated for 2 h with the vehicle or 5 μM ARP-100 (B) or ONO-4817 (C), followed by treatment with 20 μM cisplatin for 24 h. (D) IL-6 secretion in cells transfected with non-targeting siRNA ( siNT ), Mmp-9 -targeting siRNA ( siMmp-9 ), and exposed to 20 μM cisplatin for 24 h. The data are represented as the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05).

Article Snippet: For overexpression studies, the pCMV3-C-FLAG vector encoding human MMP-2 ORF (HG10082-CF, Sino Biological) was transfected into HEI-OC1 using jetPRIME Reagent (Polyplus) according to the manufacturer’s protocol.

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Matrix metalloproteinases proteolyze RAB proteins and contribute to cisplatin-induced ototoxicity

doi: 10.64898/2026.02.28.708770

Figure Lengend Snippet: A, B) Immunoblot of in vitro time course proteolysis of recombinant RAB9A at 37 °C with 100 ng MMP-2 (A) or for 30 minutes in the presence or absence of MMP-2-preferring inhibitors (B). C) In silico analysis of the top 10 MMP-2 cleavage sites in the structure of mouse RAB9A according to ProsperousPlus. D) Crystal structure of mouse RAB9A showing the predicted cleavage sites (green balls).

Article Snippet: For overexpression studies, the pCMV3-C-FLAG vector encoding human MMP-2 ORF (HG10082-CF, Sino Biological) was transfected into HEI-OC1 using jetPRIME Reagent (Polyplus) according to the manufacturer’s protocol.

Techniques: Western Blot, In Vitro, Recombinant, In Silico

Journal: bioRxiv

Article Title: Efficient cell-free evolution of RNA polymerases by droplet microfluidics

doi: 10.64898/2026.02.23.707346

Figure Lengend Snippet: (a) Optimization of elongation factor P (EF-P) and chaperone conditions. (b) Optimization of the linker length between Sso7d and SP6 RNA polymerase. (c) Crystal structure of T7 RNA polymerase initiation state (PDB ID: 2PI4). (d) Crystal structure of T7 RNA polymerase intermediate state (PDB ID: 3E2E). (e) Crystal structure of T7 RNA polymerase elongation state (PDB ID: 1H38). (f) Predicted structure of SP6 RNA polymerase by ColabFold. N-terminal domains of RNA polymerases (Residues 1-240 in SP6 RNAP, residues 1-266 in T7 RNAP) are colored in purple, other regions are colored in gray. RNAs are colored in yellow and template DNAs are colored light purple, and non-templated DNAs are in pink.

Article Snippet: Resulting gene fragment was introduced by NEBuilder HiFi DNA Assembly into the vector prepared by inverse PCR with Oligo32, Oligo33 and pET22b-FKBP-C-SP6 RNAP Lib5-3-#01 (198+) (v4)_N-SP6 RNAP-v3 (−197). pET22b-Rapa-spT7 RNAP: The gene fragment encoding N-29-1 split N-terminal T7 RNAP-FRB was amplified by PCR using Oligo34 and Oligo35 with p5-79 N-29-1 split N-terminal T7 RNAP variant-GGSGSGSS-FRB expression plasmid (#118087, Addgene) as templates.

Techniques:

Journal: bioRxiv

Article Title: Efficient cell-free evolution of RNA polymerases by droplet microfluidics

doi: 10.64898/2026.02.23.707346

Figure Lengend Snippet: (a) Crystal structure of T7 RNA polymerase elongation state (PDB ID: 1H38). Split site of previously reported spT7 RNAP is between K179 (red sphere) and K180 (blue sphere). Protein is colored in green, RNAs are colored orange, template DNAs are colored light purple, and non-templated DNAs are in pink. (b) Predicted structure of SP6 RNA polymerase by ColabFold. Split sites tried in this study are shown in red and blue spheres. (c) Rapamycin-induced spSP6 RNAP activities measured by mNG reporter assay in PURE system. The data are presented as the mean ± SD from three independent experiments. (d, e) Histograms of the fluorescence signal distribution of droplets screened for spSP6 RNAP Library 4 ( d ) or Library 5 ( e ) in FADS. Overlaid histograms are the same as or 3c. (f, g) Sequence alignment of evolved variants identified from Library 4.3 ( f ) and Library 5.3 ( g ). Residue numbers correspond to SP6 RNAP-wt (UniProt: P06221). The same residues relative to SP6 RNAP-v3 are shown in dots. ( h, i ) Rapamycin-induced spSP6 RNAPs’ activity measured by mNG reporter assay in PURE system. The data are presented as the mean ± SD from three independent experiments.

Article Snippet: Resulting gene fragment was introduced by NEBuilder HiFi DNA Assembly into the vector prepared by inverse PCR with Oligo32, Oligo33 and pET22b-FKBP-C-SP6 RNAP Lib5-3-#01 (198+) (v4)_N-SP6 RNAP-v3 (−197). pET22b-Rapa-spT7 RNAP: The gene fragment encoding N-29-1 split N-terminal T7 RNAP-FRB was amplified by PCR using Oligo34 and Oligo35 with p5-79 N-29-1 split N-terminal T7 RNAP variant-GGSGSGSS-FRB expression plasmid (#118087, Addgene) as templates.

Techniques: Reporter Assay, Fluorescence, Sequencing, Residue, Activity Assay