Dilated cardiomyopathy (DCM), which develops from myocardial inflammation, is frequently linked to a poor prognosis and fatal outcome. Experimental autoimmune myocarditis (EAM) serves as an animal model of CD4⁺ T cells-dependent acute myocarditis that is followed by the development of post-inflammatory DCM. The study's objective was to employ spatial transcriptomics to investigate how cardiac fibroblasts' transcriptional patterns changed during the acute inflammatory phase of EAM.

RNA interference is one of the common methods of studying protein functions. In recent years critical reports have emerged indicating that off-target effects may have a much greater impact on RNAi-based analysis than previously assumed. We studied the influence of Adam10 and Adam17 silencing on MC38CEA cell response to proinflammatory stimuli. Eight lentiviral vector-encoded shRNAs that reduced ADAM10 expression, including two that are specific towards ADAM17, caused inhibition of cytokine-induced Nos2 expression presumably via off-target effects. ADAM10 silencing was not responsible for this effect because: (i) CRISPR/Cas9 knockdown of ADAM10 did not affect Nos2 levels; (ii) ADAM10 inhibitor increased rather than decreased Nos2 expression; (iii) overexpression of ADAM10 in the cells with shRNA-silenced Adam10 did not reverse the effect induced by shRNA; (iv) shRNA targeting ADAM10 resulted in decrease of Nos2 expression even in ADAM10-deficient cells. The studied shRNAs influenced transcription of Nos2 rather than stability of Nos2 mRNA. They also affected stimulation of Ccl2 and Ccl7 expression. Additionally, we used vectors with doxycycline-inducible expression of chosen shRNAs and observed reduced activation of NF-κB and, to a lesser extent, AP-1 transcription factors. We discuss the requirements of strict controls and verification of results with complementary methods for reliable conclusions of shRNA-based experiments.

Premelanosomal protein (Pmel, also known as Pmel17) is the major component of melanosomal fibrils and plays a key role in melanin polymerization, making it an important factor in melanogenesis. We investigated how the absence of Pmel affects the properties of B16F10 melanoma cells. Pmel-knockout B16F10 cells were generated using CRISPR/Cas9-mediated genome editing. A viability assay revealed no significant differences between wild-type (WT) and Pmel-knockout (KO) sublines; however, melanosome maturation was impaired. In Pmel KO cells, the cell cycle was disrupted, and higher levels of reactive oxygen species (ROS) were observed compared with WT cells. Moreover, the migration capacity and tube formation of melanoma cells were increased. Tumors derived from Pmel KO cells exhibited unchanged growth kinetics but reduced melanin content, along with enhanced vascularization and oxygenation. Thus, knockout of the Pmel17 gene in melanoma cells alters pigmentation, vascularization, and oxygenation of tumors. These parameters are crucial for both tumor progression and therapeutic response.