Journal: Scientific Reports

Article Title: Caveolin-1α regulates primary cilium length by controlling RhoA GTPase activity

doi: 10.1038/s41598-018-38020-5

Figure Lengend Snippet: Cav1α controls cilium length via ROCK and Dia1. (A , B) Control and Cav1α KO cells were untreated (−) or treated with 10 µM Y27632 for 24 h and then stained for acetylated tubulin, F-actin and γ-tubulin ( A ). The scatter-plot represents the total length of cilia measured in µm; more than 700 cells were analyzed for each condition ( B ). (C) Control and DIA1 KO cells were analyzed by immunoblotting for DIA1. GAPDH was used as a loading control. (D , E) Control and DIA1 KO cells were grown for 5 days and stained for acetylated tubulin, F-actin, γ-tubulin and nuclei ( D ). The scatter-plot represents ciliary lengths measured in µm; more than 700 cells were analyzed in control and DIA1 KO cells ( E ). (F–H) Control and Cav1α KO cells were transiently transfected with the indicated constructs, then fixed and stained after 72 h for F-actin and acetylated tubulin ( F ). The scatter-plots represent the ciliary lengths measured in µm; more than 100 cells were analyzed for each condition ( G , H ). Scale bars, 5 µm. Data in B, E, G and H were pooled from at least three independent experiments and are represented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, non-significant.

Article Snippet: The sources of the antibodies to the different markers were as follows: Cav1 (mouse mAb IgG1, used at 1/2,000 for immunoblot analysis; 610406; and the rabbit polyclonal antibody used at 1/200 for immunofluorescence analysis; 610059) and DIA1 (mouse mAb IgG1, used at 1/1000 for immunoblotting; clone51/mDia1; 610484), were from BD Transduction Laboratories. γ-tubulin (mouse mAb IgG1, used at 1/500 for immunofluorescence analysis; clone GTU-88, T3559) and acetylated tubulin (mouse mAb IgG2b, used at 1/500 for immunofluorescence analysis; clone 6-11B-1; T7451) were obtained from Sigma-Aldrich.

Techniques: Staining, Western Blot, Transfection, Construct

Journal: Scientific Reports

Article Title: Caveolin-1α regulates primary cilium length by controlling RhoA GTPase activity

doi: 10.1038/s41598-018-38020-5

Figure Lengend Snippet: Model of Cav1α function in the regulation of primary cilia length. In control cells (left), Cav1α localizes in non-caveolar domains at the apical membrane of MDCK cells, where it positively regulates RhoA activity. Active RhoA subsequently promotes apical actin polymerization through its effectors ROCK1 and DIA1. Actin meshwork rearrangement regulates ciliary lengthening by controlling the access of transport vesicles to the centrosomal zone. In the absence of Cav1α (right), RhoA activation is impaired and less apical actin polymerizes, allowing the arrival of more material at the centrosome that is used to assemble longer cilia.

Article Snippet: The sources of the antibodies to the different markers were as follows: Cav1 (mouse mAb IgG1, used at 1/2,000 for immunoblot analysis; 610406; and the rabbit polyclonal antibody used at 1/200 for immunofluorescence analysis; 610059) and DIA1 (mouse mAb IgG1, used at 1/1000 for immunoblotting; clone51/mDia1; 610484), were from BD Transduction Laboratories. γ-tubulin (mouse mAb IgG1, used at 1/500 for immunofluorescence analysis; clone GTU-88, T3559) and acetylated tubulin (mouse mAb IgG2b, used at 1/500 for immunofluorescence analysis; clone 6-11B-1; T7451) were obtained from Sigma-Aldrich.

Techniques: Activity Assay, Activation Assay

Journal: BMC Medicine

Article Title: Five-year results of a treatment program for chronic hepatitis B in Ethiopia

doi: 10.1186/s12916-023-03082-4

Figure Lengend Snippet: Baseline characteristics of patients enrolled in a pilot treatment program for chronic hepatitis B in Ethiopia, 2015

Article Snippet: Co-infection with hepatitis C virus (HCV) was detected at baseline using an enzyme-linked immunosorbent assay (ELISA) method measuring anti-HCV in serum (Elisys Uno, HUMAN, Wiesbaden, Germany), whereas antibodies to hepatitis D virus (anti-HDV) was detected by ELISA in plasma samples (ETI-AB-DELTAK-2, Diasorin, Italy) and intermediate or weak positive results were confirmed by a second anti-HDV ELISA (Dia.Pro Diagnostic Bioprobes Srl, Milan, Italy).

Techniques:

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

doi: 10.1371/journal.ppat.1006007

Figure Lengend Snippet: (A) Kasumi-3 and CD34 + HPCs were efficiently infected with the NR-1 strain of HCMV. Kasumi-3 and HPCs were either mock-infected or infected with a GFP-expressing NR-1 strain at the indicated multiplicities of infection (MOIs). Two days later, the cells were analyzed for GFP expression by flow cytometry. An MOI of 5 was used for the following experiment. (B) The maintenance of the NR-1 genome, the suppression of viral IE1 and the presence of latency-associated UL138 over a 10-day time course. DNA and total RNA were isolated from Kasumi-3 cells and HPCs at various time points after infection. Viral genomic DNA was assayed by PCR, and RNA molecules encoding IE1 and UL138 were assayed by RT-PCR. In both cases, gel electrophoresis was used to detect the products of the reactions. (C) Reactivation of NR-1 virus in infected Kasumi-3 cells and HPCs. Kasumi-3 cells and HPCs were latently infected along a 10-day time course. Then, a subset of each cell population was cultured for an additional 2 days under conditions favoring lytic reactivation: Kasumi-3 cells were exposed to TPA, while HPCs were grown in reactivation medium. Following this, total RNA was extracted from the cells, and the ratio of IE1 to UL138 cDNA expression was assessed by qRT-PCR in triplicate. (D) Release of infectious progeny virions in latently infected Kasumi-3 cells and HPCs following reactivation treatment. Latently infected or mock-infected Kasumi-3 cells and HPCs were cultured under conditions favoring lytic reactivation (described above) or control conditions for 6 days, after which the cells were washed with PBS and co-cultured with HFFs for 2 days. Then, the Kasumi-3 cells were removed from the co-cultures, and the HFFs were washed with PBS and cultured for an additional 5 days for fluorescence microscopy analysis of GFP-positive plaques. (E) miR-UL148D showed robust accumulation during the establishment of experimental HCMV latency in Kasumi-3 cells. In total, 20,000 infected cells were harvested for the isolation of total RNA and DNA at each indicted time point along the 10-day time course. Viral DNA was first quantified by qPCR, and then absolute viral genomes copies were calculated by generating a standard curve. HCMV miRNAs were then assayed with a HCMV miRNA probe kit, and their levels were calculated using a standard curve. The HCMV miRNA level per virus was calculated by dividing the amount of each HCMV miRNA by the virus copy number. (F) miR-UL148D accumulated in HPCs latently infected with NR-1. HCMV miRNA levels in NR-1-infected HPCs were determined as described above. Values are shown as the mean ± SEM (n = 3). **, P<0.01.

Article Snippet: Anti-HCMV IgG and IgM antibodies in plasma were detected by ELISA using an HCMV IgG/IgM kit (DIA PRO Diagnostic Bioprobes, Milano, Italy) according to the manufacturer’s instructions.

Techniques: Infection, Expressing, Flow Cytometry, Isolation, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Virus, Cell Culture, Quantitative RT-PCR, Control, Fluorescence, Microscopy

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

doi: 10.1371/journal.ppat.1006007

Figure Lengend Snippet: (A, C) HCMV genome copies in Kasumi-3 cells (A) and CD34 + HPCs (C) infected with NR-1 or NR-1ΔmiR-UL148D. Total DNA was isolated from the infected cells at various time points after infection, and viral DNA was quantified by qPCR and normalized to cellular GAPDH. (B, D) Representative transcript levels from each class of viral genes in Kasumi-3 cells (B) and HPCs (D) infected with NR-1 or NR-1ΔmiR-UL148D. IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript). Total RNA was isolated from infected cells and assayed by RT-qPCR. Samples were assayed in triplicate, and GAPDH level was used for normalization. (E, F) Restoring miR-UL148D expression via transfection with the miR-UL148D agomir reduced HCMV genome copies (E) and IE1 (F) expression in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. The miR-UL148D agomir was added 24 hours before viral infection, and the culture media was replaced everyday with the addition of fresh agomir. DNA and total RNA were isolated from the Kasumi-3 cells at various time points after infection and quantified by qPCR and RT-qPCR, respectively. Samples were assayed in triplicate, and GAPDH level was used for normalization. Results derived from NR-1-infected Kasumi-3 cells are shown as a control. (G) Both NR-1ΔmiR-UL148D- and NR-1-infected Kasumi-3 cells produced infectious progeny. Infected Kasumi-3 cells harvested 10 days post-infection were stained with a monoclonal antibody against IE1 (clone 1B12, shown in red). GFP (green) and DAPI (blue) served as markers for lytic infection and nuclei, respectively. Infected Kasumi-3 cells were also co-cultured with HFFs. Viral plaque formation in the HFFs (shown by a GFP-positive status) was visualized by fluorescence microscopy. Images were collected using a 40x objective, and representative fields are shown for each infection. Values are shown as the mean ± SEM (n = 3). *, P<0.05. **, P<0.01.

Article Snippet: Anti-HCMV IgG and IgM antibodies in plasma were detected by ELISA using an HCMV IgG/IgM kit (DIA PRO Diagnostic Bioprobes, Milano, Italy) according to the manufacturer’s instructions.

Techniques: Infection, Isolation, Quantitative RT-PCR, Expressing, Transfection, Derivative Assay, Control, Produced, Staining, Cell Culture, Fluorescence, Microscopy

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

doi: 10.1371/journal.ppat.1006007

Figure Lengend Snippet: (A) Schematic of miR-UL148D-binding sites on IER5, with the two seed-recognizing sites marked in red. The predicted free energy of each hybrid is indicated. (B) Downregulation of IER5 and upregulation of CDC25B protein levels in Kasumi-3 cells induced by miR-UL148D mimics. The protein levels of IER5 and CDC25B in Kasumi-3 cells 48 hours after transfection with miR-UL148D mimics were assayed with western blotting. GAPDH served as an internal control. (C) Upper panel: construction of luciferase reporter that expresses the IER5 3’UTR with or without mutation of miR-UL148D-binding sites. Mutations of the seed-recognizing sites in the IER5 3’UTR are marked in red. Lower panel: firefly luciferase activity in 293T cells that were co-transfected with various luciferase reporter constructs for the IER5 3’UTR and miR-UL148D, normalized to β-gal activity. (D) Upper panel: Construction of an expression vector harboring the ORF and 3’UTR of IER5 with or without mutation of the two miR-UL148D-binding sites. The mutations in the binding sites in the IER5 3’UTR are marked in red. Lower panel: Expression levels of IER5 and CDC25B in Kasumi-3 cells that were co-transfected with wild-type IER5 (IER5 WT) or mutated IER5 (IER5 Mut) and miR-UL148D or a scrambled oligonucleotide. Protein levels were detected by Western blot analysis after a 72-h transfection. The efficiency of transfection was monitored by assaying β-gal activity. (E) Schematic representing the hypothesis for how miR-UL148D facilitates HCMV latency. Values are shown as the mean ± SEM (n = 3). **, P<0.01.

Article Snippet: Anti-HCMV IgG and IgM antibodies in plasma were detected by ELISA using an HCMV IgG/IgM kit (DIA PRO Diagnostic Bioprobes, Milano, Italy) according to the manufacturer’s instructions.

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