Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: Protein–protein interactions between stress-associated endoplasmic reticulum protein 1 (SERP1) and nonstructural protein (NS)4B. ( A ) NS4B interacts with SERP1, as shown in a membrane-base split ubiquitin yeast-two-hybrid assay. Yeast was co-transformed with the baits p-BT3 NS2B, p-BT3 NS4A, p-BT3 NS4B, and p-BT3 (vector only), and the prey pPR3-SERP1. Ten-fold serial yeast dilutions were spotted onto nonselective plates (-Trp–Leu; lacking tryptophan and leucine) and selective plates (-Trp–Leu–His–Ade; lacking tryptophan, leucine, histidine, and adenine) for the detection of protein–protein interactions. ( B ) Dengue virus type 2 (DENV-2) NS4B colocalized with SERP1 and the endoplasmic reticulum (ER) marker calnexin. The Flag-SERP1-overexpressing Huh7.5 cells were infected with DENV-2 (multiplicity of infection (MOI) = 5) and subjected to co-stain with antibodies raised against Flag, NS4B, or ER-located calnexin. At 72 h post-infection, the subcellular distributions of SERP1, NS4B, or calnexin were examined by indirect immunofluorescence assay using the corresponding antibodies. NS4B and SERP1 showed a strong colocalization (upper panel). ER-located calnexin and SERP1 (middle panel) and NS4B (lower panel) also showed colocalization. The nuclei were stained with DAPI (blue). Scale bar, 25 μm. ( C ) The colocalization analysis between SERP1, NS4B, and calnexin was quantified using Pearson’s correlation coefficients. Each counterstain was determined for 10 Huh7.5 cells in three independent experiments using the colocalization tool provided, with Leica-SP5 software. The values are shown as the average of the Pearson’s correlation coefficients in 10 cells. Error bars indicate the means ± standard errors of the mean (SEMs). ( D ) The topological scheme of the SERP1, NS4B, and NS2B complexes with selected interaction pairs in a Nano-Luc Binary Technology (NanoBiT) protein–protein interactions (PPIs) assay. ( E ) The interactions of SEPR1-NS4B and SEPR1-NS2B were determined in the HEK-293 cells using a NanoBiT complementation assay. Luminescence is expressed as the means ± SEMs from three independent experiments ( n = 3). ***, p < 0.001 (Student’s t -test). ( F ) Schematic diagram of the NS2B-HA (pLKO-AS2-NS2B-HA), NS4B-HA (pLKO-AS2-NS4B-HA), and Flag-SERP1 (pLKO-AS2-Flag-SERP1) fusion constructs. ( G ) The interaction of SEPR1-NS4B was determined in the Huh7.5 cells by co-immunoprecipitation (co-IP). The Huh7.5 cells were transfected or co-transfected with pLKO-AS2-Flag-SERP1, pLKO-AS2-NS2B-HA, and pLKO-AS2-NS4B-HA. Equal amounts of protein complexes were analyzed by immunoprecipitation using anti-Flag M2 affinity gel or anti-HA magnetic beads. The co-IP was performed with the anti-Flag antibody and anti-HA antibody.

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Protein-Protein interactions, Membrane, Ubiquitin Proteomics, Y2H Assay, Transformation Assay, Plasmid Preparation, Virus, Marker, Infection, Staining, Immunofluorescence, Software, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Magnetic Beads

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: SERP1 expressions were induced in Huh7.5 cells upon DENV-2 infection and wild-type (WT) replicon transfection. ( A ) The Huh7.5 cells were uninfected or infected with DENV-2 at an MOI = 1. The quantification of SERP1 transcripts was performed by RT-qPCR at 0, 1, 2, 3, 4, and 5 d.p.i. The relative quantitative values of the SERP1 gene were normalized to the level of β-actin. *, p < 0.05; ***, p < 0.001 (Student’s t -test). ( B ) Schematic representation of the DNA-launched DENV-2 reporter replicon pCMV-DV2Rep, which was used in a transient replicon assay. The transcriptional expression of the replicon RNA is under the control of the CMVmin promoter, and the 3′ terminus of the transcript is processed by hepatitis delta virus (HDV) ribozyme sequences. The N-terminal 102 amino acids of the C protein (C102), the Renilla luciferase gene (Rluc), the FMDV2A cleavage site, a neomycin resistance gene (Neo), an internal ribosome entry site (IRES) element, the C-terminal 24 amino acids of E (E24), the entire NS protein region (NS1 to NS5), the HDV ribozyme sequence, and the SV40 poly(A) signal sequence are indicated. The star represents the replication-defective mutant—a replicon with an inactivating mutation (Gly–Asp–Asp (GDD) to Gly–Ala–Ala (GAA) at the amino acids 662–664 of NS5) in the catalytic site of the NS5 RNA-dependent RNA polymerase (RdRp) was used as a negative control. ( C ) The Huh7.5 cells were transfected with the WT replicon or mutant replicon. The quantification of the SERP1 transcripts was performed by RT-qPCR at 0, 1, 2, 3, and 4 d.p.t. The relative quantitative values of the SERP1 gene were normalized to the level of β-actin. **, p < 0.01; ***, p < 0.001 (Student’s t -test).

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Infection, Transfection, Quantitative RT-PCR, Expressing, Control, Virus, Luciferase, Sequencing, Mutagenesis, Negative Control

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: The SERP1 overexpression inhibited DENV-2 infection, and SERP1 knockdown increased DENV-2 infection. ( A ) Western blot analysis of Flag-tagged SERP1 proteins in Huh7.5 cells expressing exogenous SERP1 and empty-vector cells. Equal amounts of lysates were incubated with anti-Flag M2 affinity gel, and the precipitates were analyzed by Western blot using the anti-Flag antibody and anti-SERP1 antibody. ( B ) The reduction in DENV-2 yields in SERP1-overexpressing cells infected with DENV-2. We established Huh7.5 cells with a stable expression of SERP1 and empty-vector cells. The cells were infected with DENV-2 at MOI = 1. The virus yields were determined at the indicated times. Infectious virus yields in the BHK21 clone 15 cells were quantified by plaque assay. Tde differences in the virus yields between the Huh7.5 cells stably expressing SERP1 and empty-vector cells at 3 or 4 d.p.i. were analyzed using Student’s t -test. *, p < 0.05. ( C ) A RT-qPCR analysis of the SERP1 mRNA expression in the empty vector HEK-293 cells and knockdown cells. shRNA-mediated knockdown of SERP1 reduces the mRNA expression in the HEK-293 uninfected cells. The expression values were normalized to the β-actin expression. The values are the means ± standard errors of the means (SEMs). ***, p < 0.001 (Student’s t -test). ( D ) The knockdown of the SERP1 cells increased the viral yields of DENV-2. We established HEK-293 cells with a SERP1 knockdown by a specific shRNA. The HEK-293 cells stably expressing shSEPR1 were infected with DENV-2 at MOI = 1. The virus yields were determined at 3 d.p.i.

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Over Expression, Infection, Knockdown, Western Blot, Expressing, Plasmid Preparation, Incubation, Virus, Plaque Assay, Stable Transfection, Quantitative RT-PCR, shRNA

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: Knockout of SERP1 by the II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system in Huh7.5 cells decreased SERP1 mRNA levels, and viral yields were significantly enhanced in the SERP1 knockout cells. ( A ) SERP1 RNA expression patterns in the SERP1 knockout sublines. The products of RT-PCR performed on RNA isolated from the parental Huh7.5 cells (SERP1 +/+ ) and SERP1 knockout sublines (SERP1 +/− and SERP1 −/− ) using the primers in SERP1 exon 1 and exon 3, which generate a 697 bp product. The SERP1 mutant allele was amplified as a 563 bp product, where the SERP1 exon 1 region was deleted. ( B ) Sequencing analysis of the parental Huh7.5 cells and SERP1 knockout cell lines from the RT-PCR product. ( C ) Detection of SERP1 mRNA levels in the parental Huh7.5 cells and SERP1 knockout sublines by qRT-PCR. The expression values are normalized to β-actin expression. ***, p < 0.001 (Student’s t -test). ( D ) Kinetics of DENV-2 replication in the parental cells and SERP1 knockout sublines. The parental Huh7.5 cells and SERP1 knockout sublines were infected with DENV-2 (MOI = 1) at the indicated times. Infectious virus yield in the BHK21 clone 15 cells was quantified by plaque assay. The differences in virus yields between the parental cells (SERP1 +/+ ) and SERP1 knockout cells (SERP1 −/− ) at 2 or 3 d.p.i. were analyzed using Student’s t -test. *, p < 0.05.

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Knock-Out, CRISPR, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Isolation, Mutagenesis, Amplification, Sequencing, Quantitative RT-PCR, Expressing, Infection, Virus, Plaque Assay

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: The effect of SERP1 on the DENV-2 replicon capacity in the Huh7.5 cells. The replication activities of the transient expression of DNA-launched mutant replicon ( A ) or WT replicon ( B ) plasmids were detected in the parental cells, stable cells overexpressing SERP1, and SERP1 knockout cells. The luciferase activity of the transfected cells was measured at 1, 2, 3, and 4 d.p.t. The error bars represent the SEMs from three independent experiments. The differences in the luciferase activity between the transfected cells at 3 or 4 d.p.t. were analyzed using Student’s t-test. ***, p < 0.001 (relative to the parental cells).

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Expressing, Mutagenesis, Knock-Out, Luciferase, Activity Assay, Transfection

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: Overexpression of NS4B improves the virus replication in the Huh7.5 cells overexpressing SERP1. ( A ) Schematic diagram of HA C-terminal fusion constructs of NS2B and NS4B. The N-terminal 2K signal peptide was deleted (△2K-NS4B-HA). ( B ) Immunoblot analysis of HA-tagged NS2B and NS4B proteins in the parental cells and SERP1-overexpressing Huh7.5 cells. All of the fragments represented in panel ( A ) were cloned in pLKO-AS2 to tag the C-terminal end of each protein. Equal amounts of lysates were incubated with anti-HA magnetic beads, and the precipitates were analyzed by Western blot using an anti-HA antibody. The parental cells ( C ) and SERP1-overexpressing Huh7.5 cells ( D ) were co-transfected with WT replicon and plasmids encoding individual viral proteins, as indicated. The cell lysates were harvested 24, 48, 72, and 96 h after transfection, and the luciferase activity of the transfected cells was measured. The replication efficiency was calculated by determining the ratio of luciferase activity obtained at 48, 72, and 96 h, to the average value obtained from all of the replicon constructs at 24 h post-transfection. The error bars represent the standard errors of the means (SEMs) from three independent experiments. *, p < 0.05; ***, p < 0.001 (Student’s t -test).

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Over Expression, Virus, Construct, Western Blot, Clone Assay, Incubation, Magnetic Beads, Transfection, Luciferase, Activity Assay

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: Hypothetical model of SERP1-mediated DENV-2 infection. ( A ) SERP1 overexpression inhibits DENV-2 viral RNA replication and titers. ( B ) DENV-2 NS4B interacts with SERP1. DENV-2 NS4B may alleviate the inhibitory effect of SERP1 on DENV-2 viral replication via the interaction of NS4B with SERP1.

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Infection, Over Expression

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: The association between SERP1 expression and clinical characteristics in SKCM patients.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: SERP1 expression in cancers. ( A ) SERP1 expression in different cancers and normal tissues in TCGA and GTEx pan-cancer data, ns, p ≥ 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001, ( B ) The SERP1 expression in SKCM and normal tissues, ( C ) The volcano plots of DEGs between high and low SERP1 expression groups, ( D ) The heatmap of top 15 up-regulated DEGs, ( E ) The heatmap of top 15 down-regulated DEGs.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Genes related to SERP1. ( A ) The heatmap of top 50 positively RNAs related to SERP1, ( B ) The heatmap of top 50 negative RNAs related to SERP1, ( C ) The PPI network of SERP1 built via STRING, ( D ) The GGI network of SERP1 built via GeneMANIA.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques:

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Functional enrichment analysis of DEGs between high and low expression of SERP1 in SKCM patients. ( A – C ) GO and KEGG pathway enrichment analyses for DEGs between High and -Low expression of SERP1 in SKCM patients. ( D ) The top 10 hub genes ranked by MCC of cytoHubba, ( E ) The top 30 hub genes ranked by MCODE.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques: Functional Assay, Expressing

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Genetic alteration and protein localization of SERP1 in SKCM patients. ( A ) Bar chart of SERP1 mutation in pan-cancers based on TCGA database. ( B ) SERP1 gene expression and mutation analysis in SKCM. ( C ) The distribution of SERP1 genomic alterations in SKCM. ( D ) The graph of the correlation between SERP1 expression and copy number alterations in SKCM. ( E ) Kaplan-Meier curve of OS in SKCM patients with altered (red) and unaltered (blue) mRNA expression of the SERP1 gene. ( F ) The representative IHC staining images from HPA database presents SERP1 expression in normal and tumor tissues.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques: Mutagenesis, Gene Expression, Expressing, Immunohistochemistry

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Correlations between SERP1 and prognosis in SKCM patients. ( A ) OS Kaplan-Meier curve for SERP1 in SKCM patients, ( B ) DSS Kaplan-Meier curve, ( C ) PFI survival Kaplan-Meier curve, ( D – O ) OS Kaplan-Meier curve of statistically significant subgroups for ( D ) Female, ( E ) male, ( F ) Age≤60, ( G ) Race white, ( H ) T stage (T2-T4), ( I ) N stage (N1-N3), ( J ) M stage (M0), ( K ) Pathologic Stage (Stage II-IV), ( L ) Radiation therapy No, ( M ) Tumor tissue site Trunk, ( N ) Melanoma ulceration Yes, ( O ) Melanoma Clark Level (Stage II-V).

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques:

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Forest plots of different clinical variables for SERP1 in SKCM patients. Forest plot of different clinical variables on OS ( A ) and DSS ( B ) by multivariate cox regression analysis.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques:

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: The prognostic nomogram for predicting OS and DSS probability. ( A ) The prognostic nomogram for predicting OS probability by the multivariable Cox regression model via the four statistically significant predictors, such as SERP1, N stage, Melanoma ulceration and Breslow depth. ( B ) The prognostic nomogram for predicting DSS probability. ( C ) The time-dependent ROC curves of OS for 1, 3, 5 year. ( D ) The time-dependent ROC curves of DSS for 1, 3, 5 year. ( E ) The calibration curve of OS for 1, 3, 5 year. ( F ) The calibration curve of DSS for 1, 3, 5 year.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques:

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: SERP1 expression is associated with different clinical variables in SKCM patients. ( A ) T classification, ( B ) Pathologic stage, ( C ) Radiation therapy, ( D ) Breslow depth, ( E ) Melanoma ulceration.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Associations of SERP1 expression and immune infiltration level in SKCM patients. ( A ) Correlation of SERP1 expression with immune infiltration level of 24 immune cell types by Spearman’s analysis. ( B ) Twenty-four types of immune cells are plotted according to different SERP1 expression levels.* p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Relationship of SERP1 expression with immune cell level in SKCM. ( A – P ) SERP1 expression showed significant positive related to infiltrating levels of T helper cells, Tcm, Tgd, Th2 cells, Macrophages, Th1 cells, B cells, aDC, T cells, CD8 T cells, Eosinophils and significant negative related to infiltrating levels of NK CD56 bright cells, NK cells, Mast cells, pDC and Th17 cells.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Correlation analysis between SERP1 and relate gene markers of immune cells in SKCM.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques: Biomarker Discovery

Journal: Aging (Albany NY)

Article Title: Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma

doi: 10.18632/aging.203594

Figure Lengend Snippet: Correlation analysis between SERP1 and immune checkpoints in SKCM.

Article Snippet: However, it is noteworthy that the expression of SERP1 protein did not show significant differences between tumor tissues and normal tissues in the Human Protein Atlas ( ).

Techniques:

Journal: Molecular Cancer

Article Title: Targeting gallbladder cancer: oncolytic virotherapy with myxoma virus is enhanced by rapamycin in vitro and further improved by hyaluronan in vivo

doi: 10.1186/1476-4598-13-82

Figure Lengend Snippet: Myxoma virus productively infects human gallbladder cancer cell lines in vitro and phosphorylated Akt expression levels in human gallbladder cancer cell lines. A . Replication over a period of 1 replication cycle was investigated using high multiplicity of infection (MOI) single-step growth curves in control permissive CV-1, GBC cell lines (GBC-SD, NOZ, SGC-996). All cells were infected with vMyx-gfp (MOI = 5), and cell lysates were collected at the indicated time points after infection. Viral titers were determined by titration in CV-1 cells. B . Replication over a period of multiple replication cycles was investigated using low MOI multi-step growth curves. CV-1, GBC-SD, NOZ and SGC-996 were infected with vMyx-gfp (MOI = 0.01). C . GFP was in visualized by fluorescence microscopy. GBC-SD, NOZ, SGC-996, a permissive glioma cell line control (U251), and a poorly-permissive murine fibroblast cell line control (NIH3T3) were infected with vMyx-gfp at an MOI = 5 and photographed 48 h after infection. D . Effects of MYXV on cell viability of GBC cell lines in vitro . E . Early viral protein was determined by M-T7 expression, and late viral protein was determined by Serp-1 expression at the indicated time points by western blot of cell lysates. F . The expression of phosphorylated Akt (Thr308) in U251, GBC-SD, NOZ, SGC-996, and NIH3T3 cells was evaluated by western blotting. G . Densitometry results of relative p-Akt expression normalized by β-Actin of each cell line in Figure F. H . GBC-SD, SGC-996, and NOZ were pretreated with Rap (20 nmol/L or 100 nmol/L) for 1 h, and then cells were infected with vMyx-gfp (MOI = 5). Levels of p-Akt (Thr308) and total Akt in cell lysates were determined by western blotting. I . Densitometry results of relative p-Akt expression normalized by β-Actin of each cell line in Figure H. FFU, fluorescent focus-forming units.

Article Snippet: Western blot examined protein expression using antibodies against MYXV M-T7 and Serp-1 (Biogen, Cambridge, MA); host p-Akt (Thr308) and Akt (Cell Signaling Technology, MA, USA); and host collagen I and IV (abcam, Cambridge, UK). β-Actin was used as the control.

Techniques: In Vitro, Expressing, Infection, Titration, Fluorescence, Microscopy, Western Blot

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet: ORF8/p62 condensates subvert ER-phagy (A) Co-precipitation analysis of GFP-FAM134B with ORF8-Strep in HEK293T. (B) Purified MBP or MBP-ORF8 was incubated with purified GST or GST-FAM134B, and analyzed the interaction between ORF8 and FAM134B by GST pull-down. (C) Immunofluorescence of cells expressing FAM134B-HA or FAM134B-HA and GFP-ORF8 or FAM134B-HA, p62-FLAG and GFP-ORF8 with anti-HA and anti-FLAG antibodies. Scale bar, 10 μm. The number of FAM134B puncta (>1 μm) in each cell was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (D) In vitro phase separation assay of GFP-FAM134B alone or GFP-FAM134B and MBP-ORF8 or GFP-FAM134B and mCherry-p62/His-UBx8 or GFP-FAM134B, MBP-ORF8 and mCherry-p62/His-UBx8. Fluorescence images of 10 μM each protein in phase separation assay buffer without PEG8000. Representative images of three independent experiments are shown. Scale bar, 10 μm. (E) GFP-ORF8 truncations were expressed as indicated with FAM134B-HA. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed using WB. (F) HeLa cells were transfected with GFP-ORF8 truncations with FAM134B-HA for 24 h, then stained with anti-HA antibody and imaged using confocal microscopy. Scale bar, 10 μm. (G and H) U2OS cells expressing mCherry-GFP-RAMP4 were transfected with p62-FLAG with or without ORF8-Strep or mutants for 24 h, then cells were starved in EBSS for 12 h and imaged using confocal microscopy (G). Scale bar, 10 μm. The number of red puncta in each cell (H) was counted from 50 cells of three independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (I and J) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h and then starved in EBSS for 12 h. Cell lysates were analyzed using WB. (K) U2OS cells expressing mCherry-Sec61B were transfected with indicated plasmids for 24 h. Cell lysates were analyzed using WB. (L) Vero-E6 cells were transfected with control or ORF8-KD plasmid for 36 h and analyzed the knockdown efficiency using WB. (M and N) Vero-E6 WT cells were transfected with control or ORF8-KD plasmid, p62 KO Vero-E6 cells were transfected with p62 EHGG-AAAA -FLAG for 12 h and then infected with SARS-CoV-2 for 24 h, then cells were stained with anti-FAM134B and anti-p62 or anti-FLAG antibodies and imaged using confocal microscopy. Scale bar, 10 μm. The number of colocalization between FAM134B and p62 droplets in each cell was counted from 20 cells of two independent experiments. Two-tailed unpaired Student’s t test, ∗∗∗∗ p < 0.0001. (O) Vero-E6 cells were transfected with ORF8-KD plasmid with or without si-ORF3a or si-Atg7 for 24 h and then infected with SARS-CoV-2 for another 24 h. Cell lysates were analyzed using WB. See also Figure S3 .

Article Snippet: Rabbit anti-SERP1 (RAMP4) pAb , Proteintech , Cat#: 17807-1-AP; RRID: AB_10597394.

Techniques: Purification, Incubation, Immunofluorescence, Expressing, Two Tailed Test, In Vitro, Fluorescence, Immunoprecipitation, Transfection, Staining, Confocal Microscopy, Control, Plasmid Preparation, Knockdown, Infection

Journal: Cell Reports

Article Title: Coronavirus subverts ER-phagy by hijacking FAM134B and ATL3 into p62 condensates to facilitate viral replication

doi: 10.1016/j.celrep.2023.112286

Figure Lengend Snippet:

Article Snippet: Rabbit anti-SERP1 (RAMP4) pAb , Proteintech , Cat#: 17807-1-AP; RRID: AB_10597394.

Techniques: Virus, Control, Recombinant, Transfection, Saline, Modification, Protease Inhibitor, Magnetic Beads, Bicinchoninic Acid Protein Assay, shRNA, Software

Journal: bioRxiv

Article Title: Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism

doi: 10.1101/2020.01.16.909200

Figure Lengend Snippet: Z-projected confocal micrographs of terminal cells expressing the membrane marker CD4::mIFP under btl-gal4 and FGFR::GFP under its own promoter (from the fTRG library). (A) Time lapse imaging of a control cell. Green arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4::mIFP and FGFR::GFP. (B) shibirets cell imaged before dynamin inactivation, after 23 and 45 minutes of inactivation, and after 30 minutes of recovery. (C-D) Single confocal planes of terminal cells expressing PH::mCherry and stained for βPS-Integrin. Green arrowheads: βPS-Integrin signal in filopodia; White arrowheads: signal at the tube membrane. The outline of the cell was traced using the PH::mCherry signal and is shown as a blue dashed line in C’ and D. (E) Quantification of FGFR::GFP fluorescence intensity from stained embryos. Number of cells analyzed: Control n=8, Restrictive temperature n=6, Recovery n=8. Significance was assessed using t-test.

Article Snippet: We used the following antibodies: mouse anti-βPS Integrin (1:200, DSHB #6G11), rat anti-ECad (1:100, DSHB #DCAD2), mouse anti-FasIII (1:200 DSHB #7G10), mouse anti-dpERK (1:200, Sigma-Aldrich #M8159), rabbit anti-Dof [1:200 (Vincent et al., 1998)] and rabbit anti-Serp (1:300, gift from Stefan Luschnig, University of Münster, Germany).

Techniques: Expressing, Marker, Imaging, Staining, Fluorescence