Journal: Life sciences

Article Title: Divergent regulation of inflammatory cytokines by mTORC1 in THP-1-derived macrophages and intestinal epithelial Caco-2 cells.

doi: 10.1016/j.lfs.2021.119920

Figure Lengend Snippet: Fig. 2. mTORC1 potentiates the LPS-mediated inflammatory response of THP-1 macrophages. a) S,R,T THP-1 macrophages were stimulated with 1 μg LPS/ml media or PBS as control for 24 h. After 24 h, conditioned media were collected, and cellular total RNA was extracted. Cytokine gene expression (TNFα, IL-6, IL- 8, IL-1β and IL-10) was measured by qRT-PCR and normalized to housekeeping gene, PPIA. Media IL-6 and IL-8 levels were measured by ELISA. Values not sharing a common letter (a,b,c,d,e next to the horizontal bars) are significantly different. One-way ANOVA followed by Tukey's multiple comparisons test; P < 0.05, n = 3–6. b) Validation of LYS6K2 efficacy (an inhibitor of p70S6K, 10 μM in DMF) as determined by a 66% decrease of S6 (Ser235/236) phosphorylation in THP-1 shScramble macrophages. Statistical significance was assessed by the Student t-test; * P < 0.05 vs. DMF control, n = 3. c) LYS6K2 (10 μM) abrogated LPS-mediated IL-6 gene expression in THP-1 shScramble macrophages. Values not sharing a common letter (a,b,c next to the horizontal bars) are significantly different. One-way ANOVA followed by Tukey's multiple comparisons test; P < 0.05, n = 3.

Article Snippet: Total RNA was isolated from cells using Aurum Total RNA Mini Kit (BioRad) that includes a DNase I incubation step.

Techniques: Control, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Phospho-proteomics

Journal: Life sciences

Article Title: Divergent regulation of inflammatory cytokines by mTORC1 in THP-1-derived macrophages and intestinal epithelial Caco-2 cells.

doi: 10.1016/j.lfs.2021.119920

Figure Lengend Snippet: Fig. 3. Conditioned media (CM) from LPS-treated THP-1 macrophages activate mTORC1 and cytokine gene expression in Caco-2 cells. a) Stably transduced THP-1 macrophages were stimulated with 1 μg LPS/ml media or PBS as control for 24 h, the CM were collected and transferred to 80% confluent regular Caco-2 cells. Caco-2 cells were cultured in the presence of THP-1 CM for 24 h. A treatment group of Caco-2 cells cultured in fresh THP-1 medium for 24 h was used as a negative control. To control for possible LPS carry over in THP-1 CM, a separate group of Caco-2 cells were cultured for 24 h in CM from THP-1 shScramble macrophages treated with PBS to which fresh LPS (1 μg LPS/ml media) was added. After 24 h, total RNA preparations of Caco-2 cells were analyzed for changes in cytokine gene expression (IL- 6, IL-8, IL-10) by qRT-PCR and normalized to housekeeping gene, PPIA. Values not sharing a common letter (a,b,c next to the horizontal bars) are significantly different. One-way ANOVA followed by Tukey's multiple comparisons test; P < 0.05, n = 3–6. b) Separate groups of 80% confluent regular Caco-2 cells were cultured for 24 h in CM from PBS or LPS-treated THP-1 shTSC2 macrophages. After 24 h, cellular proteins were extracted in modified RIPA buffer and analyzed for phos phorylated p70S6K (Thr389), p70S6K, phosphorylated p85S6K (Thr412), p85S6K, phosphorylated S6 (Ser235/236), S6, phosphorylated ERK (Thr202/Tyr204), ERK, COX-2, and β-actin by Western blotting. Statistical significance was assessed by the Student t-test; * P < 0.05, n = 3, ns = not significant.

Article Snippet: Total RNA was isolated from cells using Aurum Total RNA Mini Kit (BioRad) that includes a DNase I incubation step.

Techniques: Gene Expression, Stable Transfection, Control, Cell Culture, Negative Control, Quantitative RT-PCR, Modification, Western Blot

Journal: Life sciences

Article Title: Divergent regulation of inflammatory cytokines by mTORC1 in THP-1-derived macrophages and intestinal epithelial Caco-2 cells.

doi: 10.1016/j.lfs.2021.119920

Figure Lengend Snippet: Fig. 4. mTORC1 protects Caco-2 cells exposed to secretions of LPS-treated THP-1 shTSC2 cells. a) Stable S,R,T Caco-2 cells received the CM of PBS or LPS-treated THP-1 shTSC2 macrophages for 24 h. After 24 h, total RNA preparations were analyzed for cytokine (IL-6, IL-8, TNFα, IL-10) gene expression by qRT-PCR and normalized to housekeeping gene, PPIA, n = 3. b) Caco-2 shTSC2 cells were pre-treated with rapamycin (200 nM) for 1 h, then media was replaced with the CM from THP-1 shTSC2 treated +/− LPS and supplemented with rapamycin (200 nM). After 24 h, total RNA preparations were analyzed for cytokine (TNFα, IL-10) gene expression by qRT-PCR and normalized to housekeeping gene, PPIA, n = 3. c) Stable S,R,T Caco-2 cells were cultured in complete EMEM media until 80% confluence, at which point cellular proteins were extracted in RIPA buffer and analyzed for p-ERK (Thr202/Tyr204), ERK, p-AKT (Ser473), and AKT by Western blotting, n = 3. a-c) Values not sharing a common letter (a,b,c,d next to the bars) are significantly different. One-way ANOVA followed by Tukey's multiple comparisons test; P < 0.05.

Article Snippet: Total RNA was isolated from cells using Aurum Total RNA Mini Kit (BioRad) that includes a DNase I incubation step.

Techniques: Gene Expression, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Pain

Article Title: MicroRNA-mediated GABA Aα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats.

doi: 10.1016/j.pain.2012.09.002

Figure Lengend Snippet: Fig. 4. (A) Real-time RT-PCR analysis of differentially expressed GABAAa-1 gene in spinal dorsal horns (L6–S1) from rats with neonatal cystitis. Two different treatment protocols were followed. Tissues were examined on P60 from the unchallenged protocol and on P30 from the re-challenged protocol. The ordinate value 2DCT corresponds to the mRNA expression relative to reference gene ribosomal RNA (rRNA); data are represented as mean ± SD (n = 5 per group). (B) Western blot analysis of GABAAa-1 protein in spinal dorsal horn samples (L6–S1) from rats with neonatal cystitis. The intensity of GABAAa-1 immunoreactivity for different tissues was normalized against the intensity of b-actin expression for the same tissue. Results are expressed as means ± SD (n = 5 per group). ⁄Significant difference at p < 0.05. (C) GABAAa-1 immunostaining of differentially expressed GABAAa-1 gene in spinal dorsal horns (L6–S1) from rats with neonatal cystitis. The tissues were examined on P60 after zymosan/saline treatment at P14 to P16. The scale bar is 50 lm. The intensity of staining for 10 individual cells from each group was determined; data are presented as means ± SD. ⁄Significant difference at p < 0.001.

Article Snippet: GABAAa-1 gene expression in spinal dorsal horn neurons (L6–S1) by real-time quantitative RT-PCR Total RNA was extracted from spinal dorsal horn samples from both the unchallenged (P60) and re-challenged (P30) groups of animals using Total RNA extraction kit from Bio-Rad (#732-6830).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunostaining, Saline, Staining

Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 3. 5342191 inhibits HCoV-229E and HCoV-OC43 replication. (A) A schematic of the exper- imental protocol. Huh7 cells were infected with indicated coronavirus strains for 1 h; cells were washed and then treated with either DMSO or 5342191. Media were harvested and levels of viral RNA release into media were determined by RTqPCR. Cells were lysed for RNA extraction to measure levels of intracellular viral RNA using RTqPCR, and viral protein production via Western blotting. (B) Effect of 5342191 concentrations on HCoV-229E RNA accumulation in media (by RTqPCR) and cell viability (assessed using Alamar blue). (C) A correlation of viral RNA release and infectious virus was produced. Infected cells were treated with EC50 (1.2 µM) or EC90 (2.5 µM) doses of 5342191 for 24 h, then media were harvested, and viral RNA accumulation in media was measured by RTqPCR, infectious virus by TCID50 assay. (D) Cells were infected with the HCoV-OC43 virus at an MOI of 2 and then treated with either DMSO or 2.5 µM 5342191 for 24 h. Culture supernatants were harvested and analyzed by RTqPCR to measure the release of HCoV OC43 into the culture media. (E) Top: a schematic of the coronavirus virus genome and subgenomic RNAs generated. Shown are the positions of primer sets used to measure genomic or total viral RNAs. Bottom: infected cells were treated with DMSO or 2.5 µM 5342191 for 24 h. Extracellular viral RNA accumulation was measured by RTqPCR. Total RNA was extracted from cells and the abundance of genomic or total viral RNA (genomic and nested RNAs) was measured by RTqPCR. (F) Representative immunoblots showing the impact of 5342191 on the production of the nucleocapsid (N) protein of the HCoV-229E virus (top) and the N protein and spike (S) protein of the HCoV-OC43 virus (bottom). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Infection, RNA Extraction, Western Blot, Virus, Produced, TCID50 Assay, Generated

Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 8. 5342191 inhibits PR8 influenza A virus replication. (A) A549 cells were infected at an MOI of 2 with PR8 for 1 h, and media were removed and replaced with media containing DMSO or indicated doses of 5342191. At 24 hpi, media were harvested, and levels of viral RNA released were determined by RTqPCR. (B) To correlate the level of viral RNA released to the infectious virus, infected cells were treated with doses of 5342191 required to reduce PR8 RNA accumulation in media by 90% (EC90, 0.104 µM). Media were assessed in parallel to determine the effect of the compound addition on the level of infectious virus present as determined by TCID50 assay. (C–E) A549 cells were infected with PR8 at an MOI of 2 for 1 h, and media were removed and replaced with media containing DMSO or 0.104 µM 5342191. At 24 hpi, cells were harvested, (C) expression of viral NP or NS1 was assessed by the western blot or (D) total RNA extracted, and intracellular levels of indicated viral RNAs were determined by RTqPCR. (E) Ratio of M2 versus M1 or NS2 versus NS1 to assess the impact of 5342191 on viral RNA splicing. Shown are the results from n > 3 independent assays. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Virus, Infection, TCID50 Assay, Expressing, Western Blot

Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 9. 5342191 acts post-entry to inhibit influenza replication. A549 cells were infected at an MOI of 2 with PR8 for 1 h, and then media were replaced. Media were harvested at various times post-infection and (A) the level of viral RNA in media was determined by RTqPCR; (B,C) cells were harvested and (B) protein lysates were used for western blot to detect PR8 NP or NS1 proteins, or (C) total RNA extracted and the intracellular abundance of viral RNAs (M1, NP, and NS1) was determined by RTqPCR. (D) To assess whether 5342191 could inhibit virus replication at times after infection, cells were infected with PR8, MOI 2, for 1 h, then 0.104 µM of 5342191 was added 1, 6, 9, or 12 hpi. At 24 hpi, media was harvested, and levels of viral RNA were determined by RTqPCR. ****, p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Infection, Western Blot, Virus