Journal: PLOS Pathogens

Article Title: Transcriptional factor ATF3 impairs KSHV lytic replication by suppressing the expression of viral bZIP protein K8

doi: 10.1371/journal.ppat.1014222

Figure Lengend Snippet: (A) Schematic strategy for purification and identification of K8 binding proteins with IP assay. iSLK-RGB-K8-Flag cells were induced by doxycycline (1 μ g/ml) for 72 hours, while plasmid expressing Flag-tagged K8 was transient transfection into HEK293T.219 cells for 48 hours. Cell lysates were performed to affinity purification by immunoprecipitation with FLAG M2 beads. The purified elutes were boiled in SDS-PAGE loading buffer and then were analyzed by MS. (B) Venn diagram showing the overlaps of differentially candidate K8 binding proteins in iSLK-RGB-K8-Flag and HEK293T.219 cells. (C) HEK293T cells were transfected with K8-Flag alone, with ATF3-HA alone or with both K8-Flag and ATF3-HA. (D) HEK293T cells were transfected with ATF3-Flag alone, with K8-HA alone or with both ATF3-Flag and K8-HA. For C and D, cell lysates were immunoprecipitated with an anti-Flag antibody and were then analyzed by immunoblotting with the indicated antibodies. (E and F) Co-IP of endogenous K8 and ATF3 in iSLK-RGB-K8-Flag cells. Expression of K8 in the cells was induced by doxycycline (1 μ g/ml) for 48 hours, and cell lysates were subjected to immunoprecipitation with the anti-Flag antibody or mouse IgG control antibody (E); the anti-ATF3 antibody or rabbit IgG control antibody (F). Purified proteins, along with input samples, were subjected to immunoblotting with the indicated antibodies. (G) Colocalization of ATF3 and K8 in HEK293T cells. Following transfection with ATF3-Flag and K8-HA, cells were fixed with 4% paraformaldehyde and then stained with mouse anti-Flag antibody and rabbit anti-HA antibody, followed by incubation with goat anti-mouse IgG conjugated with Alexa Fluor 488 and goat anti-rabbit IgG conjugated with Alexa Fluor 555 to visualize the stained ATF3 and K8 proteins, respectively. Nuclei were labelled with DAPI. Cells were analyzed by Zeiss confocal microscopy and representative images with scale bars were shown.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity agarose (Sigma, A2220), 3 x Flag Peptide (Sigma, F4799), Lipofectamine 2000 (Thermo Fisher Scientific, 11668019), FuGENE HD Transfection Reagent (Promega, E2311), ATF3 agonist (MCE, HY-151923), doxycycline hyclate (Sigam, 324385), tetradecanoyl phorbol acetate (TPA, Sigma, P8139) and blasticidin (Sigma, 203350).

Techniques: Purification, Binding Assay, Plasmid Preparation, Expressing, Transfection, Affinity Purification, Immunoprecipitation, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Control, Staining, Incubation, Confocal Microscopy

Journal: PLOS Pathogens

Article Title: Transcriptional factor ATF3 impairs KSHV lytic replication by suppressing the expression of viral bZIP protein K8

doi: 10.1371/journal.ppat.1014222

Figure Lengend Snippet: (A) Truncated versions of ATF3 are shown schematically, including ATF3-bZIP (86 aa-149 aa) and the bZIP region deletion truncation of ATF3 (ATF3- Δ bZIP). (B) Defining the K8-interacting domain of ATF3. Co-IP and immunoblotting of HEK293T cells transfected with HA-tagged K8 along with plasmids expressing the empty vector, Flag-tagged ATF3 truncations or the full-length ATF3. (C) Schematic diagram of the K8 protein and its truncations, including K8-bZIP (122 aa-237 aa) and the bZIP region deletion truncation of K8 (K8- Δ bZIP, 1 aa-121 aa). (D) Defining the ATF3-interacting domain of K8. Co-IP and immunoblotting of HEK293T cells transfected with HA-tagged ATF3 along with plasmids expressing the empty vector, Flag-tagged K8 truncations or the full-length K8.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity agarose (Sigma, A2220), 3 x Flag Peptide (Sigma, F4799), Lipofectamine 2000 (Thermo Fisher Scientific, 11668019), FuGENE HD Transfection Reagent (Promega, E2311), ATF3 agonist (MCE, HY-151923), doxycycline hyclate (Sigam, 324385), tetradecanoyl phorbol acetate (TPA, Sigma, P8139) and blasticidin (Sigma, 203350).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: Transcriptional factor ATF3 impairs KSHV lytic replication by suppressing the expression of viral bZIP protein K8

doi: 10.1371/journal.ppat.1014222

Figure Lengend Snippet: (A and B) iSLK-RGB cells were treated with or without doxycycline (1 μ g/ml) at different time points as indicated. The expression kinetics of LANA, RTA and ATF3 were detected by immunoblotting (A) and the mRNA expression of ATF3 was determined by qPCR analysis (B). The protein level of LANA and ATF3 were quantified by densitometry and normalized to Tubulin level. (C) iSLK-RGB cells were stably transfected with lentiviruses containing an empty vector plasmid or a Flag-tagged ATF3 expression plasmid, named iSLK-RGB-Vector and iSLK-RGB-ATF3, respectively. The overexpression of ATF3 was detected by immunoblotting. (D) iSLK-RGB-Vector and iSLK-RGB-ATF3 cells were treated with doxycycline (1 μ g/ml) at different time points as indicated. Fluorescence microscopy images of eGFP-positive cells among iSLK-RGB-Vector and iSLK-RGB-ATF3 cells were shown. (E to G) iSLK-RGB-Vector and iSLK-RGB-ATF3 cells were induced by doxycycline (1 μ g/ml) for 72 hours, and total RNA was extracted from cells to investigate the transcriptional level of indicated genes (E); the extracellular virion DNA (F) and the intracellular viral genomic DNA (G) were extracted from cell supernatants or cell lysates to quantify the KSHV genomic DNA copy numbers by qPCR analysis. For B and E to G, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significance.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity agarose (Sigma, A2220), 3 x Flag Peptide (Sigma, F4799), Lipofectamine 2000 (Thermo Fisher Scientific, 11668019), FuGENE HD Transfection Reagent (Promega, E2311), ATF3 agonist (MCE, HY-151923), doxycycline hyclate (Sigam, 324385), tetradecanoyl phorbol acetate (TPA, Sigma, P8139) and blasticidin (Sigma, 203350).

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Over Expression, Fluorescence, Microscopy

Journal: PLOS Pathogens

Article Title: Transcriptional factor ATF3 impairs KSHV lytic replication by suppressing the expression of viral bZIP protein K8

doi: 10.1371/journal.ppat.1014222

Figure Lengend Snippet: (A) iSLK-RGB cells were transfected with control siRNA and two ATF3-specific siRNAs. The knockdown efficiency was determined by immunoblotting. (B) At 6 hours after transfection, cells were induced by doxycycline (1 μ g/ml) at different time points as indicated. Fluorescence microscopy images of eGFP-positive cells were shown. (C to E) At 6 hours after transfection, cells were induced by doxycycline (1 μ g/ml) for another 72 hours. The ATF3 and KSHV gene transcription level were analyzed by qPCR (C); the extracellular virion DNA (D) and the intracellular viral genomic DNA (E) were extracted from cell supernatants or cell lysates to quantify the KSHV genomic DNA copy numbers by qPCR analysis. For C to E, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). ****P < 0.0001.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity agarose (Sigma, A2220), 3 x Flag Peptide (Sigma, F4799), Lipofectamine 2000 (Thermo Fisher Scientific, 11668019), FuGENE HD Transfection Reagent (Promega, E2311), ATF3 agonist (MCE, HY-151923), doxycycline hyclate (Sigam, 324385), tetradecanoyl phorbol acetate (TPA, Sigma, P8139) and blasticidin (Sigma, 203350).

Techniques: Transfection, Control, Knockdown, Western Blot, Fluorescence, Microscopy

Journal: PLOS Pathogens

Article Title: Transcriptional factor ATF3 impairs KSHV lytic replication by suppressing the expression of viral bZIP protein K8

doi: 10.1371/journal.ppat.1014222

Figure Lengend Snippet: (A) Schematic diagram of K8 promoter region (from -2000 bp to -1 bp). The ATF3 binding site (from -1055 bp to -1048 bp) is marked. (B) Validation of ATF3 binding at the K8 promoter. HEK293T.219 cells were induced by TPA (20 ng/ml) for 48 hours and were subjected to ChIP assays using immunoglobulin G or the ATF3 antibody. The amounts of precipitated DNA were quantified by qPCR using primers amplifying the regions containing ATF3 binding site. (C) ATF3 suppresses K8 promoter activity in a dose-dependent manner in HEK293T.219 cells. HEK293T.219 cells were transfected with pK8 dual-reporter plasmid and increasing amounts of ATF3-expressing plasmids. At 6 hours after transfection, cells were induced by TPA (20 ng/ml) for another 48 hours and cells were harvested and lysed in lysis buffer to detect luciferase activity. (D) ATF3 has no effect on the activity of K8 promoter with deletion of ATF3 binding sites (pK8-ABS Del) in HEK293T.219 cells. HEK293T.219 cells were transfected with pK8-WT or pK8-ABS Del dual-reporter plasmids and ATF3-expressing plasmids. At 6 hours after transfection, cells were induced by TPA (20 ng/ml) for another 48 hours and cells were harvested and lysed in lysis buffer to detect luciferase activity. (E) With K8 overexpression, ATF3 suppresses K8 promoter activity in a dose-dependent manner in HEK293T cells. HEK293T cells were transfected with pK8 dual-reporter plasmid, constant amounts of K8-expressing plasmids and increasing amounts of ATF3-expressing plasmids. At 48 hours after transfection, the cells were harvested and lysed in lysis buffer to detect luciferase activity. (F) ATF3 has no effect on K8 promoter activity when K8 expression is suppressed with siRNAs in HEK293T.219 cells. HEK293T.219 cells were transfected with control siRNA and two K8-specific siRNAs. At 24 hours after siRNAs transfection, cells were transfected with pK8 dual-reporter plasmids and ATF3-expressing plasmids. At 6 hours after the plasmids-transfection, cells were induced by TPA (20 ng/ml) for another 48 hours and cells were harvested and lysed in lysis buffer to detect luciferase activity. For B to F, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significance.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity agarose (Sigma, A2220), 3 x Flag Peptide (Sigma, F4799), Lipofectamine 2000 (Thermo Fisher Scientific, 11668019), FuGENE HD Transfection Reagent (Promega, E2311), ATF3 agonist (MCE, HY-151923), doxycycline hyclate (Sigam, 324385), tetradecanoyl phorbol acetate (TPA, Sigma, P8139) and blasticidin (Sigma, 203350).

Techniques: Binding Assay, Biomarker Discovery, Activity Assay, Transfection, Plasmid Preparation, Expressing, Lysis, Luciferase, Over Expression, Control

Journal: PLOS Pathogens

Article Title: Transcriptional factor ATF3 impairs KSHV lytic replication by suppressing the expression of viral bZIP protein K8

doi: 10.1371/journal.ppat.1014222

Figure Lengend Snippet: (A and B) Effect of ATF3 on endogenous K8 expression. HEK293T.219 cells were transfected with increasing amounts of ATF3-expressing plasmids. At 6 hours after transfection, cells were induced by TPA (20 ng/ml) for another 48 hours and cells were harvested and analyzed by immunoblotting with the indicated antibodies. The protein level of K8 was quantified by densitometry and normalized to Tubulin level (A); total RNA was extracted from cells to investigate the transcriptional level of K8 (B). (C and D) Effect of ATF3 on endogenous K8 expression. iSLK-RGB-Vector and iSLK-RGB-ATF3 cells were induced by doxycycline (1 μ g/ml) for 72 hours and cells were harvested and analyzed by immunoblotting with the indicated antibodies. The protein level of K8 was quantified by densitometry and normalized to Tubulin level (C); total RNA was extracted from cells to investigate the transcriptional level of K8 (D). (E and F) Effect of wildtype ATF3 and bZIP deletion truncated ATF3 on endogenous K8 expression. HEK293T.219 cells were transfected with wildtype or bZIP deletion ATF3-expressing plasmids. At 6 hours after transfection, cells were induced by TPA (20 ng/ml) for another 48 hours and cells were harvested and analyzed by immunoblotting with the indicated antibodies. The protein level of K8 was quantified by densitometry and normalized to GAPDH level (E); total RNA was extracted from cells to investigate the transcriptional level of K8 (F). For B, D, F, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significance.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity agarose (Sigma, A2220), 3 x Flag Peptide (Sigma, F4799), Lipofectamine 2000 (Thermo Fisher Scientific, 11668019), FuGENE HD Transfection Reagent (Promega, E2311), ATF3 agonist (MCE, HY-151923), doxycycline hyclate (Sigam, 324385), tetradecanoyl phorbol acetate (TPA, Sigma, P8139) and blasticidin (Sigma, 203350).

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: Transcriptional factor ATF3 impairs KSHV lytic replication by suppressing the expression of viral bZIP protein K8

doi: 10.1371/journal.ppat.1014222

Figure Lengend Snippet: (A and B) Effect of LANA on endogenous ATF3 expression. HEK293T cells were transfected with increasing amounts of LANA-expressing plasmids. At 48 hours after transfection, cells were harvested and analyzed by immunoblotting with the indicated antibodies. The protein level of ATF3 was quantified by densitometry and normalized to Tubulin level (A); total RNA was extracted from cells to investigate the transcriptional level of ATF3 (B). (C and D) Effect of LANA on endogenous ATF3 expression. iSLK-RGB cells were transfected with control siRNA and two LANA-specific siRNAs. At 6 hours after transfection, cells were induced by doxycycline (1 μ g/ml) for another 72 hours and cells were harvested and analyzed by immunoblotting with the indicated antibodies (C); total RNA was extracted from cells to investigate the transcriptional level of ATF3 (D). (E) LANA suppresses ATF3 promoter activity in a dose-dependent manner in HEK293T cells. HEK293T cells were transfected with pATF3 dual-reporter plasmid and increasing amounts of LANA-expressing plasmids. At 48 hours after transfection, cells were harvested and lysed in lysis buffer to detect luciferase activity. (F) LANA has no effect on the activity of ATF3 promoter with deletion of LANA binding sites (pATF3-LBS Del) in HEK293T cells. HEK293T cells were transfected with pATF3-WT or pATF3-LBS Del dual-reporter plasmids and LANA-expressing plasmids. At 48 hours after transfection, cells were harvested and lysed in lysis buffer to detect luciferase activity. For B and D to F, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significance.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity agarose (Sigma, A2220), 3 x Flag Peptide (Sigma, F4799), Lipofectamine 2000 (Thermo Fisher Scientific, 11668019), FuGENE HD Transfection Reagent (Promega, E2311), ATF3 agonist (MCE, HY-151923), doxycycline hyclate (Sigam, 324385), tetradecanoyl phorbol acetate (TPA, Sigma, P8139) and blasticidin (Sigma, 203350).

Techniques: Expressing, Transfection, Western Blot, Control, Activity Assay, Plasmid Preparation, Lysis, Luciferase, Binding Assay

Journal: PLOS Pathogens

Article Title: Transcriptional factor ATF3 impairs KSHV lytic replication by suppressing the expression of viral bZIP protein K8

doi: 10.1371/journal.ppat.1014222

Figure Lengend Snippet: The cellular bZIP transcriptional factor ATF3 interacted with KSHV bZIP protein K8, leading to the repression of K8 promoter activity, which obviously decreased K8 both transcriptional and protein expression and thereby greatly impairing KSHV lytic replication. In turn, KSHV encoded transcriptional factor LANA decreased ATF3 expression through repressing its promoter, which counteracts the antiviral activity of ATF3 and ensures a complete lytic replication of KSHV.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity agarose (Sigma, A2220), 3 x Flag Peptide (Sigma, F4799), Lipofectamine 2000 (Thermo Fisher Scientific, 11668019), FuGENE HD Transfection Reagent (Promega, E2311), ATF3 agonist (MCE, HY-151923), doxycycline hyclate (Sigam, 324385), tetradecanoyl phorbol acetate (TPA, Sigma, P8139) and blasticidin (Sigma, 203350).

Techniques: Activity Assay, Expressing

Journal: bioRxiv

Article Title: ZNF423 depletion induces the integrated stress response and represents a potential vulnerability in NF1-associated MPNST

doi: 10.64898/2026.03.03.709360

Figure Lengend Snippet: (A-B) Volcano plots of whole-transcriptome differentially expressed genes (DEGs) identified by RNAseq in ST88-14 and NF90.8 cells following siRNA-mediated depletion of ZNF423 ( n =3) compared to control ( n =3). Dashed vertical lines reflect log 2 FC of −1 and 1. Dashed horizontal line reflects false discovery rate (FDR) of 0.05. Downregulated genes with log 2 FC≤-1 and -log 10 adjusted p-value above FDR threshold are denoted in blue. Upregulated genes log 2 FC≥1 and -log 10 adjusted p-value above FDR threshold are denoted in red. Raw RNAseq hit counts for ZNF423 in NF90.8 cells transfected with control siRNA ( n =3) or siZNF423 ( n =3). P-values represent unpaired, two-tailed t-tests between groups. (C) Venn diagram showing overlap of significantly downregulated DEGs common among ST88-14 and NF90.8 cell lines. Downregulated DEGs were defined as log 2 FC≥-1 and adjusted p-value of ≤0.05. (D) Dot plot of top enriched Hallmark gene signatures of shared downregulated genes (FDR≤0.05). (E) Venn diagram showing overlap of significantly upregulated DEGs common among ST88-14 and NF90.8 cell lines. Upregulated DEGs were defined as log 2 FC≥1 and adjusted p-value of ≤0.05. (F) Dot plot of top enriched Hallmark gene signatures of shared upregulated genes (FDR≤0.05). (G-H) Volcano plots of differentially expressed transcription factors and kinases in ST88-14 and NF90.8 cells following siRNA-mediated depletion of ZNF423 compared to control. (I-J) Immunoblots showing cleavage of PARP, ATF3, ATF4 and ZNF423 protein expression following siRNA-mediated depletion of ZNF423 ( n =3) compared to controls ( n =3). GAPDH serves as loading control. Protein expression was normalized to total protein and quantified using ImageJ. Error bars reflect SEM. P-values represent unpaired, two-tailed t test with Welch’s correction. ****=p-value ≤0.0001, ***=p-value≤0.001, **=p-value≤0.01, *=p-value≤0.05, ns=not significant.

Article Snippet: Immunoblots were performed using primary antibodies against ZNF423 (ABN410, Sigma-Aldrich), GAPDH (sc-365062, Santa Cruz Biotechnology), SUZ12 (3737S, Cell Signaling Technology), ATF3 (18665S, Cell Signaling Technology), ATF4 (11815S, Cell Signaling Technology), PARP (9542S, Cell Signaling Technology), and γH2A.x (9718S, Cell Signaling Technology).

Techniques: RNA sequencing, Control, Transfection, Two Tailed Test, Western Blot, Expressing

Journal: PLOS One

Article Title: Remdesivir may exacerbate ischemic acute kidney injury through molecular alterations in PGC-1α and apoptosis pathways: An in vivo study

doi: 10.1371/journal.pone.0336221

Figure Lengend Snippet: The results of western blotting (A) . The protein levels of ATF3 (B) , p-p53 (C) , p-p21 (D) , and cas3 (E) were represented in the studied groups. β-actin was used as an internal control. cas3: caspase-3, ip: intraperitoneal, I/R: ischemia/reperfusion, NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells, Rem: Remdesivir, sc: subcutaneous.

Article Snippet: Monoclonal antibodies were against PGC-1α (ab54481, Abcam), NF-κB p65 (ab16502, Abcam), Drp-1 (sc-271583), ATF3 (sc-518032), p-p53 (sc-377553), p-p21 (sc-377569), and caspase-3 (sc-7272) (Santa Cruz Biotechnology, Inc).

Techniques: Western Blot, Control