Journal: NAR Cancer

Article Title: IGF1R-targeted delivery of a bridged nucleic acid oligonucleotide-peptide conjugate for microRNA-21 inhibition in triple-negative breast cancer

doi: 10.1093/narcan/zcaf053

Figure Lengend Snippet: Inhibition of miR-21 activity with an anti-miR-21 BNA gapmer (BND5412) in MDA-MB-231 cells. ( A ) IC50 21.93 ± 1.19 nM of miR-21 inhibitor BND5412 transfected into MDA-MB-231 cells from derepression of a luciferase reporter vector with a miR-21 binding site in the 3′UTR of Renilla luciferase gene. Error bars, SD. ( B ) qPCR analysis of miR-21 and PDCD4 expression post BND5412 treatment. Error bars, SEM [** = P < .01, versus vehicle control by one-way t-test]. ( C ) De-repression of miR-21 direct target PDCD4 protein by western blot analysis. Triplicate mean ± SD.

Article Snippet: For qPCR of tumor suppressor and immune checkpoint mRNAs, 500 ng of purified total RNA were reverse transcribed with a High Capacity cDNA Reverse Transcription Kit (ThermoFisher, cat#4368814). qPCR of mRNAs was performed using TaqMan Gene Expression Assay (ThermoFisher) on an Applied Biosystems StepOnePlus RT-PCT system with the following TaqMan primers/probes to detect transcripts: GAPDH (Hs02786624_g1); PDCD4 (Hs00377253_m1); CD47 (Hs00179953_m1); PDL1 (Hs00204257_m1); PDL2 (Hs00228839_m1); and JAK2 (Hs01078136_m1).

Techniques: Inhibition, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Binding Assay, Expressing, Control, Western Blot

Journal: NAR Cancer

Article Title: IGF1R-targeted delivery of a bridged nucleic acid oligonucleotide-peptide conjugate for microRNA-21 inhibition in triple-negative breast cancer

doi: 10.1093/narcan/zcaf053

Figure Lengend Snippet: Biodistribution and therapeutic effect of BND6482 in orthotopic EMT6 allografts. ( A ) Fluorescent BND6482 distribution to EMT6 allografts in immunocompetent female Balb/c mice 2–96 h after a single IP injection of miR-21 inhibitor at 5 mg/kg. ( B ) Fluorescent BND5412 distribution to EMT6 allografts in immunocompetent female Balb/c mice 0–24 h after a single IP injection of miR-21 inhibitor at 5 mg/kg. ( C ) Syngeneic EMT6 cells are used to generate orthotopic allografts in immunocompetent female Balb/c mice. IP injection of saline vehicle control or AlexaFluor647-labeled BND6482 at 5 mg/kg once daily for 3 days inhibited tumor growth, as seen in white light images (left) or fluorescent images (right) of dissected tumors. ( D ) Tumor volumes of EMT6 orthotopic allografts stayed small after twice a week IP injection of 5 mg/kg of BND6482. Vehicle and scrambled control allowed continued growth. Error bars show mean with SEM. ** P < .01 by two-way ANOVA with Tukey’s multiple comparisons test. Significant effects were reported for Group × Day interaction. ( E ) Tumor masses of EMT6 orthotopic allografts were significantly reduced after twice a week IP injection of 5 mg/kg of BND6482. Error bars show mean with SEM. * P < .05 by one-way ANOVA with Dunnett’s multiple comparison test. ( F ) qPCR measurement of miR-21 and PDCD4 mRNA after twice-weekly IP injection of 5 mg/kg of BND6482. n = 5; error bars, SEM. * P < .05 by Mann–Whitney one-tailed t-test.

Article Snippet: For qPCR of tumor suppressor and immune checkpoint mRNAs, 500 ng of purified total RNA were reverse transcribed with a High Capacity cDNA Reverse Transcription Kit (ThermoFisher, cat#4368814). qPCR of mRNAs was performed using TaqMan Gene Expression Assay (ThermoFisher) on an Applied Biosystems StepOnePlus RT-PCT system with the following TaqMan primers/probes to detect transcripts: GAPDH (Hs02786624_g1); PDCD4 (Hs00377253_m1); CD47 (Hs00179953_m1); PDL1 (Hs00204257_m1); PDL2 (Hs00228839_m1); and JAK2 (Hs01078136_m1).

Techniques: Injection, Saline, Control, Labeling, Comparison, MANN-WHITNEY, One-tailed Test

Journal: bioRxiv

Article Title: ZCCHC4 Promotes Translation of Replication-dependent Histone mRNAs by Recruiting Cytoplasmic eIF3 complex

doi: 10.1101/2025.06.21.660898

Figure Lengend Snippet: a, Western blot analysis of ZCCHC4 in the subcellular fractions of HeLa and PLC/PRF/5 cells. Tubulin was used as a cytoplasmic marker, Histone H1.2 and Histone H3 as the nuclear markers. b, Identification of ZCCHC4 interacting partners. ZCCHC4 complex was purified from ZCCHC4-FH stable cells using immunoaffinity purification. Eluted proteins were separated by SDS-PAGE and analyzed by silver staining. c, ZCCHC4 associates with the multiple subunits of the eIF3 complex (highlighted by red dots) in mass spectrometry analysis. d, Western blot confirmed that eIF3H and PDCD4 are the binding proteins of ZCCHC4. e, GO analysis of ZCCHC4-associated proteins identified by mass spectrometry. f, g, Global protein synthesis in ZCCHC4 knockout (KO) and control cells was measured by puromycin assay in Hela cells ( f, left) and PLC/PRF/5 ( f, right) cells. Quantification of protein expression levels is shown in ( g ). h-j, Polysome profiling were showed the change of global translation after ZCCHC4 knockout in Hela cells ( h, up) and PLC/PRF/5 cells ( i ). Western blot confirms fractionation integrity in Hela cells using 40S (RPS6) and 60S (RPL14) ribosomal markers, with GAPDH as a ribosome-free loading control ( h, down). The experiment was repeated three times, and the ratio of poly- to mono- (AUC) was statistically analyzed ( j ). Note: g , j , One-way ANOVA with Tukey’s test. * P <0.05, *** P <0.001. Data were presented as mean ± SD (n=3).

Article Snippet: The corresponding antibodies included anti-ZCCHC4 antibody with 1:1000 dilution (Abcam, ab209901), anti-RPS6 antibody with 1:1000 dilution (Proteintech, 66886-1-Ig), anti-RPL14 antibody with 1:1000 dilution (Proteintech, 14991-1-AP), anti-CDKN1A antibody with 1:1000 dilution (Proteintech, 10355-1-AP), anti-puromycin antibody with 1:1000 dilution (ABclonal, A21205), anti-H2B antibody with 1:2000 dilution (PTM-1007), anti-H2A antibody with 1:2000 dilution (ABclonal, A3692), anti-H2AZ antibody with 1:2000 dilution (Proteintech, 16441-1-AP), anti-HistoneH1.2 antibody with 1:1000 dilution (Proteintech, 19649-1-AP), anti HistoneH3 antibody with 1:2000 dilution (ABclonal, A22348), anti-α-Tubulin antibody with 1:3000 dilution (Proteintech, 80762-1-RR), anti-EIF3H antibody with 1:1000 dilution (Proteintech, 11310-1-AP), anti-EIF3A antibody with 1:1000 dilution (Proteintech, 26178-1-AP), anti-PDCD4 antibody with 1:1000 dilution (Proteintech, 12587-1-AP), anti-CyclinB1 antibody with 1:1000 dilution (ABclonal, A19037), anti-GAPDH antibody with 1:5000 dilution (Proteintech, 10494-1-AP), anti-β-actin antibody with 1:3000 dilution (SAB Signalway Antibody, 52901), and anti-Flag antibody with 1:2000 dilution (Sigma, F1804).

Techniques: Western Blot, Marker, Purification, Immunoaffinity Purification, SDS Page, Silver Staining, Mass Spectrometry, Binding Assay, Knock-Out, Control, Expressing, Fractionation