Journal: Frontiers in immunology

Article Title: LncRNA Nostrill promotes interferon-γ-stimulated gene transcription and facilitates intestinal epithelial cell-intrinsic anti- Cryptosporidium defense.

doi: 10.3389/fimmu.2024.1397117

Figure Lengend Snippet: FIGURE 3 Molecular insights into Nostrill and STAT1 interaction in regulating ISG transcription. (A) Evidence of physical interaction between Nostrill and p65 in intestinal epithelial cells. IEC4.1 cells were exposed to a 5 ng/mL dose of IFN‐g for 2h, followed by RNA immunoprecipitation (RIP) analysis utilizing an anti-Stat1 antibody. The presence of Nostrill was quantified through qRT-PCR and normalized against the percent input. Significance (*p < 0.05) was determined when compared to the control between the indicated groups. (B–D) Recruitment of Nostrill to ISG promoter sites following IFN‐g treatment. IEC4.1 cells were exposed to a 5 ng/mL dose of IFN‐g for 2h, and subsequent ChIRP analysis was carried out using pool of probes specific to Nostrill, along with designed PCR primer sets targeting Stat1 binding sites of Igtp, Gadd45g, and iNos. A pool of Lacz probes was used as negative control. Significance levels (*p < 0.05) were assessed in comparison to the control. (E–G) Influence of Nostrill siRNA knockdown on Stat1 recruitment to Igtp, Gadd45g, and iNos promoter sites upon IFN‐g stimulation. IEC4.1 cells were transfected with siR_Nostrill or siR_Ctrl for 24h, then exposed to IFN‐g for 2h, followed by ChIP analysis using anti-p65 antibody and designed PCR primer sets. Statistical significance (*p < 0.05) was determined in comparison to siR_Ctrl. Data are presented as means ± SEM with “n” of 3.

Article Snippet: For silencing Igtp and Gadd45g, siRNA duplexes were purchased from Santacruz Biotechnology with the catalogue numbers sc-41792 and sc-37419, respectively.

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Control, Binding Assay, Negative Control, Comparison, Knockdown, Transfection

Journal: Frontiers in immunology

Article Title: LncRNA Nostrill promotes interferon-γ-stimulated gene transcription and facilitates intestinal epithelial cell-intrinsic anti- Cryptosporidium defense.

doi: 10.3389/fimmu.2024.1397117

Figure Lengend Snippet: FIGURE 5 Evidence for Nostrill-mediated enhancement of anti-Cryptosporidium defense via IFN-g-induced autophagy in intestinal epithelial cells. (A, B) Assessment of autophagy in Nostrill knockdown cells. (A) IEC4.1 cells were exposed to IFN-g for 24h, followed by Western blot analysis to evaluate the autophagy marker LC3II (14kDa), with beta-actin (31kD) serving as a reference protein. A similar analysis was conducted on Nostrill-knocked down cells exposed to IFN-g. (B) Quantification of LC3II levels in Nostrill knockdown cells was performed via qPCR. (C) Impact of Nostrill associated genes on autophagy. A combination of siRNAs was used to knockdown Igtp, iNos, and Gadd45g (referred to as siR_ISGs) in IEC4.1 cells, followed by treatment with 10 ng/ml of IFN-g for 24h. The level of LC3II marker was quantified through qRT-PCR. (D-F) Impact of Nostrill associated ISGs in defending C. parvum through autophagy. (D) IEC cells were treated with 10 ng/ml of IFN g for 24h and then infected with C. parvum for 8h. Level of Cp18s and LC3II were measured by qRT-PCR. (E, F) IEC4.1 cells were treated with siR_ISGs (260 nM), which consisted of a pool of siRNAs targeting Igtp (80 nM), iNos (60 nM), and Gadd45g (120 nM), or a scrambled siR_Ctrl at 260 nM. Subsequently, cells were treated with 10 ng/ml of IFN-g for 24h and then infected with C. parvum for 8h. (E) Cp18s, representative of C. parvum burden, and (F) LC3II levels as an indicator of autophagy were quantified using qRT-PCR. Statistical significance was calculated using t-test. Significance levels (*p < 0.05). Data are presented as means ± SEM with “n” of 3.

Article Snippet: For silencing Igtp and Gadd45g, siRNA duplexes were purchased from Santacruz Biotechnology with the catalogue numbers sc-41792 and sc-37419, respectively.

Techniques: Knockdown, Western Blot, Marker, Quantitative RT-PCR, Infection

Journal: Frontiers in immunology

Article Title: LncRNA Nostrill promotes interferon-γ-stimulated gene transcription and facilitates intestinal epithelial cell-intrinsic anti- Cryptosporidium defense.

doi: 10.3389/fimmu.2024.1397117

Figure Lengend Snippet: FIGURE 3 Molecular insights into Nostrill and STAT1 interaction in regulating ISG transcription. (A) Evidence of physical interaction between Nostrill and p65 in intestinal epithelial cells. IEC4.1 cells were exposed to a 5 ng/mL dose of IFN‐g for 2h, followed by RNA immunoprecipitation (RIP) analysis utilizing an anti-Stat1 antibody. The presence of Nostrill was quantified through qRT-PCR and normalized against the percent input. Significance (*p < 0.05) was determined when compared to the control between the indicated groups. (B–D) Recruitment of Nostrill to ISG promoter sites following IFN‐g treatment. IEC4.1 cells were exposed to a 5 ng/mL dose of IFN‐g for 2h, and subsequent ChIRP analysis was carried out using pool of probes specific to Nostrill, along with designed PCR primer sets targeting Stat1 binding sites of Igtp, Gadd45g, and iNos. A pool of Lacz probes was used as negative control. Significance levels (*p < 0.05) were assessed in comparison to the control. (E–G) Influence of Nostrill siRNA knockdown on Stat1 recruitment to Igtp, Gadd45g, and iNos promoter sites upon IFN‐g stimulation. IEC4.1 cells were transfected with siR_Nostrill or siR_Ctrl for 24h, then exposed to IFN‐g for 2h, followed by ChIP analysis using anti-p65 antibody and designed PCR primer sets. Statistical significance (*p < 0.05) was determined in comparison to siR_Ctrl. Data are presented as means ± SEM with “n” of 3.

Article Snippet: For silencing Igtp and Gadd45g, siRNA duplexes were purchased from Santacruz Biotechnology with the catalogue numbers sc-41792 and sc-37419, respectively.

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Control, Binding Assay, Negative Control, Comparison, Knockdown, Transfection