Journal: Scientific Reports

Article Title: Epigenetics, N-myrystoyltransferase-1 and casein kinase-2-alpha modulates the increased replication of HIV-1 CRF02_AG, compared to subtype-B viruses

doi: 10.1038/s41598-019-47069-9

Figure Lengend Snippet: Silencing the NMT1 or CKIIα genes blocked Tat.AG but not Tat.B-induced HIV-1 transactivation in U38 ( A – C ) and TZM-bl ( D – F ) cells. ( A , D ) RT-PCR confirmation of NMT1, NMT2, CKIIα and CKIIβ genes silencing. ( B , E ) Western blot confirmation of NMT1, NMT2, CKIIα and CKIIβ genes silencing. Control: non-transduced cells. S: cells transduced with scrambled shRNA. ( C , F ) Silencing the NMT1 or CKIIα genes blocked Tat.AG-induced increase in CAT enzyme levels ( C ) and luciferase activity ( F ), but had no effect on Tat.B-induced viral transactivation. Silencing the NMT2 or CKIIβ genes had no effect on Tat.AG or Tat.B-induced viral transactivation. *P < 0.05, **P < 0.01, # P < 0.0001, compared to cells treated with similar concentrations of scrambled (S) shRNA and Tat proteins. Errors bars represent SD.

Article Snippet: Gene knockdown was monitored by Western blot and RT-PCR, using the following Santa Cruz primers: NMT1 (h)-PR (sc-61132-PR), NMT2 (h)-PR (sc-61134-PR), CKIIα′ (h)-PR (sc-38963-PR), CKIIβ (h)-PR (sc-29916-PR), and β-Actin (h)-PR (sc-108069-PR) per manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Transduction, shRNA, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Epigenetics, N-myrystoyltransferase-1 and casein kinase-2-alpha modulates the increased replication of HIV-1 CRF02_AG, compared to subtype-B viruses

doi: 10.1038/s41598-019-47069-9

Figure Lengend Snippet: Silencing the NMT1 gene blocked the infection of human MDM by HIV-1 CRF02_AG isolates, but had no effect on clade-B HIV-1 infection. Silencing of NMT1, NMT2, CKIIα and CKIIβ genes in primary human MDM was confirmed by RT-PCR ( A ) and Western blot analyses ( B ). For panels A and B , controls are non-transduced MDM. “S” are MDM transduced with scrambled shRNA. Following NMT1 and NMT2 gene silencing, MDM were infected with CRF02_AG or clade-B HIV-1 isolates (3 different isolates for each subtype, each isolate tested in triplicate) and RT activity measured from day-5 to day-21 p.i. ( C , D ) RT activity in MDM with NMT1 gene silenced. ( E , F ) RT activity in MDM with NMT2 gene silenced. For panels C – F , controls are non-HIV infected (mock-infected) MDM, “S” are MDM transduced with scrambled shRNA; “sh” are MDM transduced with NMT1 or NMT2 shRNA. **P < 0.01; # P < 0.0001, compared to infected MDM treated with similar concentrations of scrambled shRNA (S).

Article Snippet: Gene knockdown was monitored by Western blot and RT-PCR, using the following Santa Cruz primers: NMT1 (h)-PR (sc-61132-PR), NMT2 (h)-PR (sc-61134-PR), CKIIα′ (h)-PR (sc-38963-PR), CKIIβ (h)-PR (sc-29916-PR), and β-Actin (h)-PR (sc-108069-PR) per manufacturer’s instructions.

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transduction, shRNA, Activity Assay

Journal: Scientific Reports

Article Title: Epigenetics, N-myrystoyltransferase-1 and casein kinase-2-alpha modulates the increased replication of HIV-1 CRF02_AG, compared to subtype-B viruses

doi: 10.1038/s41598-019-47069-9

Figure Lengend Snippet: Tat.AG and HIV-1 CRF02_AG, but not Tat.B or clade-B viruses, induced NMT1 expression in primary human monocytes and MDM. ( A – D ) Exposure of human monocytes ( A , C ) and MDM ( B , D ) to Tat.AG induced a dose-dependent ( A , B ) and time-dependent ( C , D ) increase in NMT1 expression and secretion. ( A , B ) cells exposed to Tat proteins for 24 h; ( C , D ) Tat proteins used at 100 ng/ml. ( E – H ) Exposure of human monocytes ( E , F ) and MDM ( G , H ) to HIV-1 CRF02_AG isolates induced a time-dependent increase in NMT1 expression and secretion. *P < 0.05, **P < 0.01, ***P < 0.001, # P < 0.0001. P-values are in comparison to HI-Tat or Tat.B ( A – D ), or clade-B viruses ( E – H ).

Article Snippet: Gene knockdown was monitored by Western blot and RT-PCR, using the following Santa Cruz primers: NMT1 (h)-PR (sc-61132-PR), NMT2 (h)-PR (sc-61134-PR), CKIIα′ (h)-PR (sc-38963-PR), CKIIβ (h)-PR (sc-29916-PR), and β-Actin (h)-PR (sc-108069-PR) per manufacturer’s instructions.

Techniques: Expressing, Comparison

Journal: PloS one

Article Title: MicroRNA-99 family members suppress Homeobox A1 expression in epithelial cells.

doi: 10.1371/journal.pone.0080625

Figure Lengend Snippet: Figure 1. MiR-100-mediated down-regulation of its target genes. The miR-100 mimic and negative control microRNA were introduced into the 1386Ln (A) and HaCaT cells (B). qRT-PCR was performed to assess the expression of mTOR, HOXA1, CTDSPL, NMT1, TMEM30A and SMARCA5. Data represents at least 3 independent triplicate experiments with similar results. * indicates p,0.05. doi:10.1371/journal.pone.0080625.g001

Article Snippet: For functional analysis, hsa-miR-99a, hsa-miR-99b, hsa-miR-100 and non-targeting microRNA mimic (Dharmacon), and gene specific siRNAs for mTOR, HOXA1, CTDSPL, NMT1, TMEM30A, SMARCA5 and Bcl-2 (Santa Cruz Biotechnology) were transfected into the cells using DharmaFECT Transfection Reagent 1 as described previously [20,21].

Techniques: Negative Control, Quantitative RT-PCR, Expressing

Journal: PloS one

Article Title: MicroRNA-99 family members suppress Homeobox A1 expression in epithelial cells.

doi: 10.1371/journal.pone.0080625

Figure Lengend Snippet: Figure 2. Correlation of miR-100 level and the expression of its target genes in HNSCC. The level of miR-100 and the expression of mTOR, HOXA1, CTDSPL, NMT1, TMEM30A and SMARCA5 were assessed by qRT-PCR in 10 HNSCC cell lines. Potential correlation of the miR-100 level with the expression of mTOR (A), HOXA1 (B), CTDSPL (C), NMT1 (D), TMEM30A (E), and SMARCA5 (F) was assessed, and the Pearson’s correlation coefficient (r) was calculated. doi:10.1371/journal.pone.0080625.g002

Article Snippet: For functional analysis, hsa-miR-99a, hsa-miR-99b, hsa-miR-100 and non-targeting microRNA mimic (Dharmacon), and gene specific siRNAs for mTOR, HOXA1, CTDSPL, NMT1, TMEM30A, SMARCA5 and Bcl-2 (Santa Cruz Biotechnology) were transfected into the cells using DharmaFECT Transfection Reagent 1 as described previously [20,21].

Techniques: Expressing, Quantitative RT-PCR

Journal: PloS one

Article Title: MicroRNA-99 family members suppress Homeobox A1 expression in epithelial cells.

doi: 10.1371/journal.pone.0080625

Figure Lengend Snippet: Figure 3. The effect of miR-100 target genes on proliferation and cell migration. 1386Ln (A and B) and HaCaT cells (C and D) were transfected with either negative control microRNA, miR-100 mimic, negative control siRNA, or specific siRNAs against mTOR, HOXA1, CTDSPL, NMT1, TMEM30A or SMARCA5. Proliferation (A and C) and cell migration (B and D) were measured as described in the Material and Methods section. Data represents at least 3 independent triplicate experiments with similar results. * indicates p,0.05. doi:10.1371/journal.pone.0080625.g003

Article Snippet: For functional analysis, hsa-miR-99a, hsa-miR-99b, hsa-miR-100 and non-targeting microRNA mimic (Dharmacon), and gene specific siRNAs for mTOR, HOXA1, CTDSPL, NMT1, TMEM30A, SMARCA5 and Bcl-2 (Santa Cruz Biotechnology) were transfected into the cells using DharmaFECT Transfection Reagent 1 as described previously [20,21].

Techniques: Migration, Transfection, Negative Control