Journal: Heliyon

Article Title: Mycoplasma hyorhinis infection promotes TNF-α signaling and SMAC mimetic-mediated apoptosis in human prostate cancer

doi: 10.1016/j.heliyon.2023.e20655

Figure Lengend Snippet: M. hyorhinis promotes TNF-α secretion from PCa cells. (A), relative TNF-α secretion levels in M. hyorhinis-contaminated PCa cells (PC3-cM and C4–2B-cM) and mycoplasma-free cells; multiple cancer cells (PC3, C4–2B, DU145, LNCaP, MDA-MB-231, MCF-7, MCF10A, HeLa, MG-63), HDMEC, HF, and DPC via ELISA. (B), experimental design. The parental PCa cells (PC3–P and C4–2B–P) were infected with M. hyorhinis (3 × 107 CFU, 5 MOI) for 2 passages and then passaged twice a week for 6 weeks without further infection (PC3-M and C4–2B-M). For PC3-MF and C4–2B-MF cells, 4 weeks after infection, M. hyorhinis was eliminated in cell cultures for 3 weeks. Seven weeks after infection, in vitro assays and analyses were performed using these PCa cells. (C), relative TNF-α secretion levels in the parental (PC3–P and C4–2B–P) and M. hyorhinis-infected (PC3-M and C4–2B-M) PCa cells. (D), relative TNF-α secretion levels in PC3-M and C4–2B-M and previously infected PCa cells after elimination of M. hyorhinis (PC3-MF and C4–2B-MF). M. hyrorhinis infection was confirmed by Western blotting using specific anti-M. hyorhinis (P70 surface antigen) antibody. βactin was used as a loading control. See full images in Supplementary Figure S4. All results represent mean ± SD values from triplicate assays, and the experiments were repeated three times. **p < 0.0001. HDMEC, human dermal microvascular endothelial cells; HF, human primary fibroblasts; DPC, human primary dental pulp cells; CFU, colony-forming units; MOI, multiplicity of infection.

Article Snippet: Human primary fibroblasts (HF) [ ], human primary dental pulp cells (DPC) [ ], and human cancer cell lines (PC3, C4–2B, DU145, LNCaP, MDA-MB-231, MCF-7, MCF10A, HeLa, MG-63; American Type Culture Collection (ATCC), Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carsbad, CA) supplemented with 10 % FBS and 1 % penicillin/streptomycin.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, In Vitro, Western Blot

Journal: Cancer Discovery

Article Title: Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers

doi: 10.1158/2159-8290.CD-24-0027

Figure Lengend Snippet: Translating RMC-6236 activity in NSCLC. A, Efficacy of RMC-6236 on Kras G12C , Kras G12D , Kras G12V , Kras G12A , Kras G13D , or Kras Q61H -driven autochthonous lung tumors in immunocompetent mice. A pool of lentiviral cDNA vectors encoding each oncogenic Kras variant was delivered intratracheally to the lungs of each mouse, and 13 weeks after tumor growth, mice were treated with RMC-6236 at 20 mg/kg po qd for 3 weeks prior to analysis. 95% confidence intervals are shown. B, Efficacy of RMC-6236 and adagrasib in the LUN055 NSCLC PDX model with KRAS G12C allele copy-number gain. Immunoblot Western analyses (left) of RAS and KRAS protein levels in NCI-H358 ( KRAS G12C/WT , NSCLC), LU99 ( KRAS G12C/WT , NSCLC), NCI-H2122 ( KRAS G12C/G12C , NSCLC), and LUN055 ( KRAS G12C/WT , NSCLC) xenograft tumors. Relative copy-number (middle) of KRAS WT or KRAS G12C in LUN055 xenograft tumors ( n = 2) were determined by ddPCR and normalized to ACTB . LUN055 xenograft tumor-bearing mice were treated with vehicle or RMC-6236 at 25 mg/kg po qd or adagrasib at 100 mg/kg po qd for 24 to 28 days ( n = 3 per group, right). Mean tumor volumes of each group were plotted over the course of treatment. Dotted line indicates the initial average tumor volume. Error bars, SEM. C, Efficacy of RMC-6236 in the intracranially implanted LU99-Luc ( KRAS G12C/WT , NSCLC) xenograft model ( n = 8 per group). RMC-6236 was dosed at 25 mg/kg daily for 21 days. Images of bioluminescence in individual mice were shown. Bioluminescence of ROI in vehicle control and RMC-6236 groups were compared by two-way repeated-measures ANOVA at day 21 (**, P < 0.01). Results were shown as mean ± SEM. D, Antitumor activity of RMC-6236 and the combination with anti–PD-1 (clone RMP1-14, rat IgG2a) following repeated administration in BALB/c mice bearing the murine colon carcinoma eCT26 ( Kras G12C/G12C ) shown as individual tumor growth curves ( n = 10 per group). Graphs indicate the number of complete regressions per injected mice. RMC-6236 and anti–PD-1 treatment started on day 17 after implantation. RMC-6236 treatment was stopped at day 31 after implantation and anti–PD-1 at day 35 after implantation. E, Antitumor activity of RMC-6236 following repeated administration in NSG mice bearing the murine colon carcinoma eCT26 ( Kras G12C/G12C ) shown as individual tumor growth curves ( n = 10 per group). Graphs indicate the number of complete regressions per injected mice. RMC-6236 treatment started on day 16 after implantation. F, Immune cell composition (CD8 + and CD4 + T cells, Ly6C + and Ly6G + myeloid-derived suppressor cells and M2 macrophages) in murine colon carcinoma eCT26 syngeneic tumors ( Kras G12C/G12C ) represented as percentage of CD45 + cells and expression of cell-surface markers on viable, CD45 − large cells (assessed as tumor cells) 24 hours post 4 days of treatment with vehicle or RMC-6236 at 25 mg/kg po qd n = 3 biological replicates/group represented as mean; *, P < 0.05; **, P < 0.01; ns, nonsignificant by two-sided Student t test.

Article Snippet: Probes and primers for the following genes were included in the multiplexed ddPCR: KRAS WT [dPCR Mutation Detection Assay KRAS Wild-Type for p.G12C, Human (apexbio, AA100902-WT)], KRAS G12C [dPCR Mutation Detection Assay KRAS Mutant for p.G12C, Human (apexbio, AA100902-MU)], and ACTB ( ACTB probe, 5′-Cy5-ATTGCCGACAGGATGCAGAAGGA-3′; ACTB primer F, 5′-GACATCCGCAAAGACCTGTA-3′; ACTB primer R, 5′-GGAAAGACACCCACCTTGAT-3′).

Techniques: Activity Assay, Variant Assay, Western Blot, Control, Injection, Derivative Assay, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A negatively regulates the TGF-β/SMAD signaling pathway. (A) The effect of KDM6A overexpression on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in Huh7 and LM3 cells was (A) determined by western blotting and (B) semi-quantified. The effect of KDM6A knockdown on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in YY-8103 and SNU-398 was (C) determined by western blotting and (D) semi-quantified. ** P<0.01 and *** P<0.001 vs. vector or SCR. KDM6A, lysine demethylase 6A; TGF-β, transforming growth factor-β; p, phosphorylated; PCNA, proliferating cell nuclear antigen; SCR, scrambled; sh, short hairpin RNA; ns, not significant.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Over Expression, Expressing, Western Blot, Plasmid Preparation, shRNA

Journal: Microbiology Spectrum

Article Title: Influenza A virus interferes with respiratory syncytial virus in mice and reconstituted human airway epithelium

doi: 10.1128/spectrum.03187-24

Figure Lengend Snippet: Kinetics of interferon (IFN) expression in the lungs of mice infected with respiratory syncytial virus (RSV) and influenza A virus (IAV). Interferon (IFN)-α and -β (type I) and IFN-λ2/3 (type III) expressions were measured in lung homogenates by RT-ddPCR daily after RSV ( A ) and IAV ( B ) infection. Results are expressed as the mean of the ratio of IFN mRNAs over that of the 18S housekeeping gene (both in copies per µL) ± standard error of the mean of three mice per group from a single experiment.

Article Snippet: Droplet digital PCR reactions were performed using QX200 ddPCR EvaGreen SuperMix (Bio-Rad Laboratories, Ltd., Mississauga, ON, Canada).

Techniques: Expressing, Infection, Virus

Journal: Microbiology Spectrum

Article Title: Influenza A virus interferes with respiratory syncytial virus in mice and reconstituted human airway epithelium

doi: 10.1128/spectrum.03187-24

Figure Lengend Snippet: Expression of interferon (IFN) in the lungs of mice infected with respiratory syncytial virus (RSV), influenza A virus (IAV), or both viruses. Mice received intranasally the vehicle (mock), RSV, IAV, or both viruses simultaneously or sequentially with a 1- [Seq(1d)] or 4-day [Seq(4d)] interval. IFN-α ( A ), IFN-β ( B ), and IFN-λ2/3 ( C ) mRNAs were determined in lung homogenates by RT-ddPCR on day 4 after the last infection. Results are expressed as the ratio of IFN mRNAs over that of the 18S housekeeping gene (both in copies per mL) ± standard error of the mean of four to eight mice per group from three independent experiments. *, compared with single or simultaneous infection; # , compared with sequential coinfection; * , # P ≤ 0.05; ** , ## P ≤ 0.01; *** , ### P ≤ 0.001.

Article Snippet: Droplet digital PCR reactions were performed using QX200 ddPCR EvaGreen SuperMix (Bio-Rad Laboratories, Ltd., Mississauga, ON, Canada).

Techniques: Expressing, Infection, Virus

Journal: Microbiology Spectrum

Article Title: Influenza A virus interferes with respiratory syncytial virus in mice and reconstituted human airway epithelium

doi: 10.1128/spectrum.03187-24

Figure Lengend Snippet: Interferon-stimulated gene (ISG) expression in nasal human airway epithelia (HAEs) infected with respiratory syncytial virus (RSV), influenza A virus (IAV), or both viruses. Nasal HAEs were infected with RSV, IAV, or both viruses simultaneously or sequentially with a 1- [Seq(1d)] or 4-day [Seq(4d)] interval. Non-infected (NI) HAEs were used in parallel. Expression of four ISGs [OAS1 ( A ), IFITM3 ( B ), ISG15 ( C ), and MxA ( D )] was measured in cell lysates by RT-ddPCR on day 5 after the last infection, except for the Seq(1d) groups (4 days after the second viral challenge). Results are expressed as the mean of the ratio of ISG mRNAs over that of 18S housekeeping gene (both in copies per µL) ± standard error of the mean of three to five nasal HAE inserts from two independent experiments. *, compared with single or simultaneous infection; # , compared with sequential coinfection; * , # P ≤ 0.05; ** , ## P ≤ 0.01; *** , ### P ≤ 0.001.

Article Snippet: Droplet digital PCR reactions were performed using QX200 ddPCR EvaGreen SuperMix (Bio-Rad Laboratories, Ltd., Mississauga, ON, Canada).

Techniques: Expressing, Infection, Virus