Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: KTN1-AS1 interacts with RBBP4 in the nucleus. ( A ) The subcellular location of KTN1-AS1 in esophageal squamous cell carcinoma (ESCC) cell lines. ( B ) RNA pull-down assay was performed in Kyse150 and Kyse170 cells, and the RNA-related proteins were determined with SDS-PAGE gel and coomassie brilliant blue staining. Original gel image was presented in Supplementary Fig. A. ( C ) Western blot assay was performed to detect the specific association between RBBP4 and KTN1-AS1 in Kyse150 and Kyse170 cells. Original western blots were presented in Supplementary Fig. B, with blots cut prior to hybridization with antibodies. ( D ) RIP assay showed the interaction between KTN1-AS1 and RBBP4 in Kyse150 and Kyse170 cells. ( E ) The regulation effect of KTN1-AS1 on RBBP4 expression was detected by qRT-PCR and western blot. Original western blots were presented in Supplementary Fig. C, with blots cut prior to hybridization with antibodies. ( F ) The relevance between KTN1-AS1 and RBBP4 expression was predicted by the GEPIA database. ( G ) The relative expression of RBBP4 in 182 tumor samples compared with 286 normal samples obtained from the Gene Expression Profiling Interactive Analysis (GEPIA) database. ( H ) The expression levels of RBBP4 in ESCC and the corresponding normal tissues. ( I ) The expression levels of RBBP4 in ESCC cell lines. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Pull Down Assay, SDS Page, Staining, Western Blot, Hybridization, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: KTN1-AS1 relates to epithelial-to-mesenchymal transition (EMT) process by interacting with RBBP4 and HDAC1 to silence E-cadherin expression. ( A ) The mRNA expression levels of E-cadherin , N-cadherin , Vimentin , and MMP2 after KTN1-AS1 overexpression and knockdown. ( B ) The regulatory effect of KTN1-AS1 on protein levels of E-cadherin, N-cadherin, Vimentin, and MMP2 was detected by western blot. Original western blots were presented in Supplementary Fig. D, with blots cut prior to hybridization with antibodies. ( C ) Inhibition of RBBP4 increased the expression level of E-cadherin and partially reversed the regulation effect of KTN1-AS1 on the expression level of E-cadherin in Kyse150 and Kyse170 cells. ( D ) Co-IP assay was performed to examine the RBBP4-HDAC1 interaction in the groups with KTN1-AS1 overexpression and inhibition in Kyse150 and Kyse170 cells. Original western blots were presented in Supplementary Fig. E, with blots cut prior to hybridization with antibodies. ( E ) RIP assay showed the interaction between KTN1-AS1 and HDAC1 in Kyse150 and Kyse170 cells. ( F ) After transfection of pcDNA3.1-NC or pcDNA3.1-KTN1-AS1 for 12–24 h in Kyse150 and Kyse170 cells, then cells with pcDNA3.1-KTN1-AS1 were treated with or without 300 nM Trichostatin A (TSA) for additional 48 h, the mRNA expression of E-cadherin was detected by qRT-PCR method. ( G ) ChIP-qPCR was performed to detect the enrichment of ac-H3 in the promoter region of E-cadherin after overexpression and knockdown of KTN1-AS1 in Kyse150 cells. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Hybridization, Inhibition, Co-Immunoprecipitation Assay, Transfection, Quantitative RT-PCR, ChIP-qPCR

Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: RBBP4 partially reverses the biological function of KTN1-AS1 on esophageal squamous cell carcinoma (ESCC) cells. ( A , B ) MTS and clone formation assays were performed to analyze the cell proliferation ability after co-transfected with pcDNA3.1-KTN1-AS1 and si-RBBP4 in Kyse150 cells. ( C , D ) Wound healing and transwell invasion assays were conducted to explore the migration and invasion ability after co-transfected with pcDNA3.1-KTN1-AS1 and si-RBBP4 in Kyse150 cells. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Transfection, Migration

Journal: Journal of Virology

Article Title: The crosstalk between ubiquitination and GlcNAcylation of CHAF1A regulates HIV-1 latency and reactivation

doi: 10.1128/jvi.01518-25

Figure Lengend Snippet: Multi-omics reveals ubiquitin-O-GlcNAc crosstalk in CAF-1-dependent HIV-1 latency. ( A ) J-Lat 10.6 cells were treated with DMSO or TNFα, 24 h later, the cells were lysed and immunoprecipitated with anti-CHAF1A antibody. Western blot was performed with CHAF1A and GAPDH antibodies. ( B ) Silver staining of immunoprecipitation samples following TNF-α activation. ( C ) Combined Sankey and bubble plot showing GO analysis of mass spectrometry-identified ubiquitination/glycosylation-related genes, where the Sankey diagram displays protein-GO term relationships and the bubble plot indicates gene ratios and significance levels (−log10 P -value). ( D ) KEGG pathway analysis of mass spectrometry data. ( E and F ) GSEA plots showing the pathways of differentially expressed genes involved in metabolic regulation, including fructose and mannose metabolism and glycolysis/gluconeogenesis.

Article Snippet: The CHAF1A ubiquitination was detected by western blotting using anti-HA (M180-3, MBL), anti-Flag (PM020, MBL), and anti-GAPDH (10494-1-AP, Proteintech) antibodies.

Techniques: Biomarker Discovery, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Silver Staining, Activation Assay, Mass Spectrometry, Glycoproteomics

Journal: Journal of Virology

Article Title: The crosstalk between ubiquitination and GlcNAcylation of CHAF1A regulates HIV-1 latency and reactivation

doi: 10.1128/jvi.01518-25

Figure Lengend Snippet: OGT/OGA balance CHAF1A stability via ubiquitin-O-GlcNAc antagonism. ( A ) Illustration of the HBP and O-GlcNAcylation mechanism. ( B ) Dynamic regulation of O-GlcNAcylation by OGT and OGA enzymes. ( C ) J-Lat 10.6 cells were infected with sgRNA targeting OGT (sgOGT) or OGA (sgOGA), with scrambled sgRNA as control (sgNC). Cells were lysed and analyzed by western blot using CTD110.6 (O-GlcNAc), anti-CHAF1A, and anti-GAPDH antibodies. ( D ) CHAF1A interacts with both OGT and OGA in Jutkat cells. Jurkat cells were harvested, and cell lysates were immunoprecipitated with anti-CHAF1A antibody or IgG control, followed by western blot analysis. GAPDH served as loading control for input samples. ( E ) Endogenous OGT and OGA interact with CHAF1A in Jurkat cells. Jurkat cells were harvested and cell lysates were immunoprecipitated with anti-OGT antibody, with IgG as control. GAPDH served as loading control for input samples. ( F ) Western blot analysis for ubiquitin on CHAF1A immunoprecipitated from J-Lat 10.6 and HEK293T cells in the presence/absence of OGA inhibitor PUGNAc. The cells were treated with/without MG132 for proteasome inhibition. Cells were treated with 50 µM PUGNAC (an inhibitor of OGA) for 48 h, followed by an additional 6-h treatment with 20 µM MG132 prior to harvest. ( G ) HEK293T cells transfected with HA-tagged CHAF1A or GFP, and then treated with PUGNAc or not. The O-GlcNAcylated CHAF1A ubiquitination was analyzed by Co-IP with anti-HA beads followed by western blot with CTD110.6. GAPDH was shown as a loading control.

Article Snippet: The CHAF1A ubiquitination was detected by western blotting using anti-HA (M180-3, MBL), anti-Flag (PM020, MBL), and anti-GAPDH (10494-1-AP, Proteintech) antibodies.

Techniques: Ubiquitin Proteomics, Infection, Control, Western Blot, Immunoprecipitation, Inhibition, Transfection, Co-Immunoprecipitation Assay

Journal: Journal of Virology

Article Title: The crosstalk between ubiquitination and GlcNAcylation of CHAF1A regulates HIV-1 latency and reactivation

doi: 10.1128/jvi.01518-25

Figure Lengend Snippet: TFD targets CHAF1A through ubiquitin-O-GlcNAc interplay. ( A ) Schematic representation of the high-throughput sequencing method. ( B ) Chemical structure of TFD. ( C and D ) Degradation efficiency of TFD to CHAF1A in HEK293T cells at the indicated concentrations. ( E ) HEK293T cells were treated with TFD at 10 µM for 48 h, followed by fixed and immunofluorescence. ( F ) Cell viability was assessed using CellTiter-Glo assay after 48-h treatment with indicated concentrations of TFD. HEK293T cells maintained >80% viability at all concentrations. Data represent mean ± SEM from three independent experiments. ( G ) J-lat10.6 cells treated with PBS or 10 µM TFD for 48 h were harvested. The level of O-GlcNAc was determined in total cell lysates by western blotting with CTD110.6 antibody. ( H ) J-Lat 10.6 cells were treated with 10 µM TFD and MG132 (20 µM, 6 h) for 48 h. Immunoprecipitation with anti-CHAF1A antibody followed by Western blot with ubiquitin antibody. ( I ) Jurkat cells were lysed with or without 10 µM TFD treatment, and O-GlcNAc-modified proteins were enriched by sWGA. Input and sWGA pull-down (IP) fractions were analyzed by immunoblotting using CTD110.6 (O-GlcNAc), anti-CHAF1A, and GAPDH antibodies.

Article Snippet: The CHAF1A ubiquitination was detected by western blotting using anti-HA (M180-3, MBL), anti-Flag (PM020, MBL), and anti-GAPDH (10494-1-AP, Proteintech) antibodies.

Techniques: Ubiquitin Proteomics, Next-Generation Sequencing, Immunofluorescence, Glo Assay, Western Blot, Immunoprecipitation, Modification

Journal: Journal of Virology

Article Title: The crosstalk between ubiquitination and GlcNAcylation of CHAF1A regulates HIV-1 latency and reactivation

doi: 10.1128/jvi.01518-25

Figure Lengend Snippet: E3 ligase MIB1 triggers CHAF1A ubiquitination by TFD. ( A ) E3 ligases predicted by the UbiBrowser database. ( B ) HEK293T cells were co-transfected with co-transfected with pEGFP-CHAF1A-C1, pcDNA3.1-CFP, and siRNAs (siNC [control], siITCH, siMDM2, siFBXW7, siNEDD4, and siMIB1), then treated with TFD. GFP fluorescence intensity is used for quantitative drug degradation. ( C ) HEK293T cells were co-transfected with CHAF1A-HA and increased amounts of MIB1-Flag (up) or NEDD4-Flag (down). At 24 h post-infection, the cells were lysed, and western blot was performed with HA, FLAG, and GAPDH antibodies. ( D ) HEK293T cells were co-transfected with CHAF1A-HA and GFP-Flag (control), NEDD4-Flag, or MIB1-Flag. Then the cells were treated with MG132 for proteasome inhibition. At 48 h post-infection, cells were lysed, and western blot was performed with HA, FLAG, and GAPDH antibodies. ( E ) J-Lat10.6 cells with MIB1 knockout (sgMIB1) or control sgRNA (sgNC) were treated with the indicated concentrations of TFD for 24 h. CHAF1A protein levels were analyzed by western blot, with GAPDH as a loading control.

Article Snippet: The CHAF1A ubiquitination was detected by western blotting using anti-HA (M180-3, MBL), anti-Flag (PM020, MBL), and anti-GAPDH (10494-1-AP, Proteintech) antibodies.

Techniques: Ubiquitin Proteomics, Transfection, Control, Fluorescence, Infection, Western Blot, Inhibition, Knock-Out

Journal: Journal of Virology

Article Title: The crosstalk between ubiquitination and GlcNAcylation of CHAF1A regulates HIV-1 latency and reactivation

doi: 10.1128/jvi.01518-25

Figure Lengend Snippet: Targeting OGT/OGA destabilizes CHAF1A to reactivate latent HIV-1. ( A ) J-Lat 10.6 cells were treated with OSMI-4 (an inhibitor of OGT), DMSO was used as a control. 48 h after treatment, the cells were harvested, and the percentage of GFP-positive cells was measured by flow cytometry. ( B ) J-Lat 10.6 cells were pre-treated with OGA inhibitor PUGNAc for 24 h, followed by OGT inhibitor OSMI-4 at indicated concentrations for 48h. HIV-1 reactivation was measured by the percentage of GFP-positive cells using flow cytometry. ( C )Cell viability was evaluated by CellTiter-Glo assay after 48-h treatment with different concentrations of OSMI-4 (25, 50, 100, and 200 µM). Data are presented as mean ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA. ( D ) J-Lat 10.6 cells were transduced with sgRNA targeting OGT (sgOGT) or a non-targeting control (sgNC). After selection, HIV-1 reactivation was determined by flow cytometry for GFP expression. ( E and F ) J-Lat 10.6 cells were treated with increased concentration of TFD, PBS was used as a control. 24 h after treatment, the cells were harvested, the percentage of GFP-positive cells was measured by flow cytometry ( E ), and the cells were lysed, and western blot was performed with CHAF1A and β-actin antibodies ( F ). ( G ) OGT inhibitor reduces global O-GlcNAcylation in J-Lat 10.6 cells. J-Lat 10.6 cells were treated with OGT inhibitor at 0, 50, or 100 µM for 48 h. Whole cell lysates were analyzed by western blot using an anti-O-GlcNAc antibody to assess global protein O-GlcNAcylation. GAPDH was used as a loading control. ( H ) TFD treatment reduces global O-GlcNAcylation levels in a dose-dependent manner. J-Lat 10.6 cells were treated with increasing concentrations of TFD (0, 5, 10, and 20 µM) for 48 h. Whole cell lysates were analyzed by western blot using anti-O-GlcNAc antibody (CTD110.6) to assess global protein O-GlcNAcylation. GAPDH was used as a loading control. ns, not significant ( P > 0.05); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The CHAF1A ubiquitination was detected by western blotting using anti-HA (M180-3, MBL), anti-Flag (PM020, MBL), and anti-GAPDH (10494-1-AP, Proteintech) antibodies.

Techniques: Control, Flow Cytometry, Glo Assay, Transduction, Selection, Expressing, Concentration Assay, Western Blot

Journal: Journal of Virology

Article Title: The crosstalk between ubiquitination and GlcNAcylation of CHAF1A regulates HIV-1 latency and reactivation

doi: 10.1128/jvi.01518-25

Figure Lengend Snippet: TFD reactivates latent HIV-1 and links CHAF1A to aging. ( A ) The procedure of HIV-1 reactivation in primary CD4 + T cells of PLWH. ( B ) Resting CD4 + T cells were isolated from HIV-1-infected individuals; 1 day later, the cells were treated with PBS (control), anti-CD3/CD28, or TFD. The cells were harvested for HIV-1 RNA quantitative detection 2 days after treatment. The corresponding statistical results were presented with three individual donors. ( C ) Schematic overview of the TZM-bl viral outgrowth assay (qVOA) using peripheral blood and rectal tissue samples from people with HIV (PWH) undergoing ART. ( D ) Quantification of HIV-1 reactivation by luciferase assay in samples from five donors. Cells were treated with DMSO (MOCK), anti-CD3/CD28, or TFD at 50 µM (TFD50) or 100 µM (TFD100). Data are shown as mean ± SD; statistical significance was determined by two-way ANOVA with Tukey’s multiple comparison test. ( E–G ) Analysis in healthy controls (HC): ( E ) CHAF1A expression is significantly increased in the old group; ( F ) OGA expression is significantly decreased in the old group; ( G ) strong negative correlation between CHAF1A and OGA expression. ( H–J ) Analysis in HIV+ individuals: ( H ) CHAF1A expression shows an increasing trend in the old group; ( I ) OGA expression is significantly decreased in the old group; ( J ) moderate negative correlation between CHAF1A and OGA expressions. ns, not significant ( P > 0.05); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The CHAF1A ubiquitination was detected by western blotting using anti-HA (M180-3, MBL), anti-Flag (PM020, MBL), and anti-GAPDH (10494-1-AP, Proteintech) antibodies.

Techniques: Isolation, Infection, Control, Viral Outgrowth Assay, Luciferase, Comparison, Expressing

Journal: Journal of Virology

Article Title: The crosstalk between ubiquitination and GlcNAcylation of CHAF1A regulates HIV-1 latency and reactivation

doi: 10.1128/jvi.01518-25

Figure Lengend Snippet: Schematic representation of CHAF1A-mediated HIV-1 latency and reactivation. Aberrant glucose metabolism in HIV-infected cells leads to the accumulation of UDP-GlcNAc, which participates in the O-GlcNAcylation of CHAF1A, thereby stabilizing its expression. This stabilization promotes HIV-1 latency and the establishment of deep viral reservoirs. Exogenous administration of TFD removes the O-GlcNAc modification from CHAF1A and mediates its degradation via the E3 ubiquitin ligase MIB1, thereby releasing the suppression on the HIV-1 genome and reactivating the latent viral reservoir.

Article Snippet: The CHAF1A ubiquitination was detected by western blotting using anti-HA (M180-3, MBL), anti-Flag (PM020, MBL), and anti-GAPDH (10494-1-AP, Proteintech) antibodies.

Techniques: Infection, Expressing, Modification, Ubiquitin Proteomics