Journal: Journal of Virology

Article Title: Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication

doi: 10.1128/JVI.00217-17

Figure Lengend Snippet: Effect of CRT0066101 on HRV 2C and viral RNA expression following infection. (A) HeLa cells were pretreated for 1 h with increasing concentrations of CRT0066101, followed by infection with HRV16 at an MOI of 20 for 1 h. Following a 6-h replication period, RNA was extracted from cell lysates, and the viral RNA level was quantified by qRT-PCR and normalized to the 18S RNA level. The results show the means (±SEM) from three independent experiments, each performed in duplicate. The “input” level (dotted line) reflects the viral RNA that was cell bound at the start of the replication cycle. (B) HeLa cells were pretreated for 1 h with increasing concentrations of CRT0066101, followed by infection with HRV16 at an MOI of 20 for 1 h. Cell extracts were prepared following a 6-h replication period and analyzed by Western blotting with antibodies to autophosphorylation residue S916 of PKD1, PKD1, HRV 2C, and LB1. Controls are as follows: uninfected cells (lane 1), PDBu-treated cells (lane 2), and vehicle control-treated cells (lane 3). Cells treated with CRT0066101 at concentrations from 0.1 to 20 μM are shown in lanes 4 to 13. (C) HBECs cells were pretreated for 1 h with increasing concentrations of CRT0066101, followed by infection with HRV1B at an MOI of 20 for 1 h. Cell extracts were prepared following a 6-h replication period and analyzed by Western blotting with antibodies against HRV 2C and LB1. Uninfected cells are shown in lane 1, and vehicle control-treated cells are shown in lane 2. (D) The effect of CRT0066051 and XX-50 on HRV 2C protein expression was analyzed by using the same protocol as the one described above for panel B. Results from each experiment shown in panels B to D are representative of data from three independent repeats. (E) In order to confirm the pharmacodynamic effect of the inhibitors on cells, HeLa cells were treated for 8 h with CRT0066101 and XX-050 at 5 μM and CRT0066051 at 10 μM, followed by analysis by confocal microscopy. The Golgi apparatus was revealed by staining with an anti-GM130 antibody, followed by staining with an anti-rabbit antibody coupled to Alexa Fluor 546 (bar = 10 μm).

Article Snippet: CRT0066101 was described previously ( ) and was purchased for this study from Tocris, with a purity of 99.9%.

Techniques: RNA Expression, Infection, Quantitative RT-PCR, Western Blot, Residue, Control, Expressing, Confocal Microscopy, Staining

Journal: Journal of Virology

Article Title: Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication

doi: 10.1128/JVI.00217-17

Figure Lengend Snippet: Effect of PKD inhibitors on picornavirus replication. (A) HeLa cells were infected with HRV16 at an MOI of 20, and replication was allowed to proceed for 6 h in the presence of increasing concentrations of CRT0066101, CRT0066051, or XX-050. Viral replication was determined as the endpoint titer (TCID 50 ). (B) HeLa cells and HBECs were infected with HRV1B at MOIs of 1 and 5, respectively, and replication was allowed to proceed for 6 h in the presence of increasing concentrations of CRT0066101. Viral replication was determined as the endpoint titer (TCID 50 ). (C and D) HeLa cells were infected with PV (C) and BHK21 cells were infected with FMDV (D) at an MOI of 20, replication was allowed to proceed for 6.5 h in the presence of increasing concentrations of CRT0066101, and viral replication was determined as the endpoint titer (TCID 50 ). All the virus titer graphs show the means (±SEM) of data from three independent experiments. Differences between infected DMSO-treated cells and drug-treated cells were estimated by using one-way ANOVA with Dunnett's post hoc test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (E) HBECs and BHK21 and HeLa cells were incubated with increasing concentrations of CRT0066101 for 10, 8.5, and 8 h, respectively, and cell viability was determined as described above. (F) TEER was measured on HBECs grown in ALI cultures and treated with CRT0066101 at increasing concentrations for 48 and 72 h. Results in panels E and F show the means (±SEM) of data from three independent experiments.

Article Snippet: CRT0066101 was described previously ( ) and was purchased for this study from Tocris, with a purity of 99.9%.

Techniques: Infection, Virus, Incubation

Journal: Journal of Virology

Article Title: Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication

doi: 10.1128/JVI.00217-17

Figure Lengend Snippet: Effect of CRT0066101 on viral entry. (A) HeLa cells were infected with HRV16 (MOI of 20) for 6 h, and CRT0066101 (5 μM) was added at the following different time points: 1 h before infection (−1), during the 1-h virus infection period (0), and every hour after the time of virus adsorption (+1, +2, +3, +4, and +5). Cells extracts were prepared at the end of the 6-h replication period and analyzed by Western blotting with anti-2C and anti-LB1 antibodies. Uninfected cells and DMSO-treated cells infected for 6 h were used as controls. Data from a representative experiment from three independent repeats are shown. (B) 2C Western blots were scanned as described in Materials and Methods and quantified by using ImageJ. The mean 2C/LB1 ratio (±SEM) is shown as a percentage of the value for the DMSO control from three independent experiments. (C) In parallel, virus was extracted from the cell lysates, and viral replication was quantified by endpoint titer determination (TCID 50 ). Results are the means (±SEM) of data from three independent experiments, each done in triplicate. Differences between DMSO-treated cells (−) and the rest of the conditions in both panels B and C were estimated by using one-way ANOVA with Dunnett's post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D) HeLa cells were grown on coverslips and pretreated with DMSO or CRT0066101 at 5 μM for 1 h at 37°C. Human transferrin conjugated to Alexa Fluor 488 was added at a 75-μg/ml final concentration, and cells were incubated at 37°C for 1 h in the presence of DMSO or CRT0066101. Cells were stained with an anti-GM130 antibody followed by an anti-mouse antibody coupled to Alexa Fluor 546 and analyzed by confocal microscopy. Transferrin quantification is shown as the mean fluorescence intensity (MFI) from multiple low-power images. For each experiment, images from five different low-power fields with approximately 250 cells per field were quantified by using ImageJ. Data shown are the means (±SEM) from three independent experiments. No statistically significant difference between DMSO- and CRT0066101-treated cells was found by performing a two-tailed t test. (E) High-power images of HeLa cells showing internalized transferrin (green) and Golgi membrane (GM130) (red) staining from a representative experiment (bar = 10 μm).

Article Snippet: CRT0066101 was described previously ( ) and was purchased for this study from Tocris, with a purity of 99.9%.

Techniques: Infection, Virus, Adsorption, Western Blot, Control, Concentration Assay, Incubation, Staining, Confocal Microscopy, Fluorescence, Two Tailed Test, Membrane

Journal: Journal of Virology

Article Title: Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication

doi: 10.1128/JVI.00217-17

Figure Lengend Snippet: Effect of CRT0066101 on interferon signaling. (A) Effect of CRT0066101 on STAT1 phosphorylation at residue Y701 in HeLa cells infected with HRV16. Cells were either untreated (lane 1) or treated with 30 U/ml IFN-β for 15 min (lane 2), the DMSO vehicle (lane 3), or increasing concentrations of CRT0066101 for 1 h, followed by a 6-h replication period. Cell extracts were prepared and analyzed by Western blotting with antibodies to pSTAT1 Y701, STAT1, HRV 2C, and LB1. Data shown are representative of results from three independent experiments. (B and C) To determine the effect of CRT0066101 on ISG expression, RNA was extracted from HRV16-infected HeLa cells after 20 h of culture in the presence of the DMSO vehicle or 1 μM, 2 μM, or 3.5 μM CRT0066101. UV-inactivated virus was included as a control. Viral replication was confirmed by measuring the levels of HRV16 RNA (HRV) (B) and OAS mRNA (C) as a representative ISG. The results are the means (±SEM) of data from four independent experiments, each performed in duplicate. Differences between infected DMSO-treated cells and infected CRT0066101-treated cells were determined by one-way ANOVA with Dunnett's post hoc analysis. *, P < 0.05; ****, P < 0.0001. (D) HeLa cells were pretreated for 1 h with the DMSO vehicle or CRT0066101 at 5 μM, followed by stimulation with IFN-β (30 U/ml) for 4, 6, and 8 h in the presence of DMSO or CRT0066101. Cells were harvested, and RNA was extracted and processed for qRT-PCR. The OAS mRNA level was measured and normalized to the 18S RNA level. The results are the means (±SEM) of data from three independent experiments, each performed in duplicate. Differences between DMSO-treated and CRT0066101-treated cells at each time point were determined by two-way ANOVA with Sidak's post hoc test. *, P < 0.05; **, P < 0.01.

Article Snippet: CRT0066101 was described previously ( ) and was purchased for this study from Tocris, with a purity of 99.9%.

Techniques: Phospho-proteomics, Residue, Infection, Western Blot, Expressing, Virus, Control, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication

doi: 10.1128/JVI.00217-17

Figure Lengend Snippet: Effect of blocking of type I interferon receptor signaling on CRT0066101. In order to determine if a blockade of the type I interferon receptor (IFNAR2) influenced the ability of CRT0066101 to inhibit viral replication, HeLa cells were left untreated (lane 1) or pretreated with DMSO (lanes 2 to 4) or 5 μM CRT0066101 (lane 5 to 7) for 1 h. Cells were subsequently infected with HRV16 for 1 h (lanes 2 to 7), and replication was allowed to proceed for a further 4 h. During the viral infection and replication periods, cells were treated with a blocking antibody to IFNAR2 alone (lane 3) or with an isotype-matched control antibody alone (lane 4) or cotreated with CRT0066101 and a blocking antibody (lane 6) or CRT0066101 with an isotype control (lane 7). As additional controls, cells were treated with 30 U/ml of IFN-β for 4 h (lanes 8 to 10), with no antibody (lane 8), with anti-IFNAR2 (lane 9), or with an isotype control antibody (lane 10). Cell extracts were prepared and analyzed by Western blotting with antibodies to pSTAT1 Y701, STAT1, HRV 2C, and LB1. The pSTAT1 Y701 blot is shown at high and low exposures, and data shown are from a representative experiment from three independent repeats.

Article Snippet: CRT0066101 was described previously ( ) and was purchased for this study from Tocris, with a purity of 99.9%.

Techniques: Blocking Assay, Infection, Control, Western Blot

Journal: Nature communications

Article Title: Multiple structures of RNA polymerase II isolated from human nuclei by ChIP-CryoEM analysis.

doi: 10.1038/s41467-025-59580-x

Figure Lengend Snippet: Fig. 2 | Details of the EC-SPT4/5-ELOF1-SPT6 structure. a Domain architectures of the elongation factors. The known domains are indicated. b Close-up view of ELOF1 (purple). c Close-up view around the upstream DNA. d Close-up view of the SPT5 KOW2, KOW3, KOWx, and KOW4 domains. Each KOW domain is indicated with dotted circles. SPT6 is omitted for clarity. e Close-up view of the KOW5 domain. f The SPT6-SPT5 KOW3-RPB4/7 stalk interaction. g The SPT5 KOW1-SPT6

Article Snippet: Themembraneswerewashedwith TBS-T (20mMTris-HCl (pH 7.5), 137mM NaCl, and 0.1% Tween-20) three times and then incubated overnight with anti-FLAG M2 antibody (Sigma # F3165, 1:3000), anti-SPT5 antibody (Proteintech#16511-1-AP, 1:1000), antiSPT6 antibody (Cell Signaling #15616, 1:2000), anti-PAF1 antibody (Cell Signaling #12883, 1:300), anti-RTF1 antibody (Proteintech #12170-1-AP, 1:1000), anti-MED17 antibody (Proteintech#11505-1-AP, 1:2000), or anti-XPB antibody (Cell Signaling #8746, 1:300) in Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO #NKB-101).

Techniques:

Journal: Autophagy

Article Title: Regulation of N-degron recognin-mediated autophagy by the SARS-CoV-2 PLpro ubiquitin deconjugase

doi: 10.1080/15548627.2024.2442849

Figure Lengend Snippet: Reagents used in the investigation.

Article Snippet: pEGFP N1 Ub-CRT , Addgene , 69619; Marta Hallak lab.

Techniques: Recombinant, Diagnostic Assay, DC Protein Assay, Transfection, Mutagenesis, Plasmid Preparation, Gel Extraction, Purification, Cell Culture, shRNA

Journal: Journal of Clinical Medicine

Article Title: The Role of Soluble Low-Density Lipoprotein Receptor-Related Protein-1 in Obstructive Sleep Apnoea

doi: 10.3390/jcm10071494

Figure Lengend Snippet: Calreticulin (CALR) concentrations between the control and obstructive sleep apnoea (OSA) group. Data are presented with median, minimum and maximum values.

Article Snippet: Plasma sLRP-1 and CALR levels were measured using commercially available ELISA kits (Human Low Density Lipoprotein Receptor Related Protein 1 ELISA Kit from Bioassay Technology Laboratory, Shanghai Korain Biotech Co. Ltd. Inc, Shanghai, China (Catalogue number: E2298Hu)); Human Calreticulin (CRT) ELISA Kit from Cusabio Technology Llc., Houston, TX, USA (Catalogue number: CSB-E09787h)).

Techniques: Control

Journal: Journal of Clinical Medicine

Article Title: The Role of Soluble Low-Density Lipoprotein Receptor-Related Protein-1 in Obstructive Sleep Apnoea

doi: 10.3390/jcm10071494

Figure Lengend Snippet: Calreticulin (CALR) concentrations between the severity groups of obstructive sleep apnoea (OSA). Data are presented with median, minimum and maximum values.

Article Snippet: Plasma sLRP-1 and CALR levels were measured using commercially available ELISA kits (Human Low Density Lipoprotein Receptor Related Protein 1 ELISA Kit from Bioassay Technology Laboratory, Shanghai Korain Biotech Co. Ltd. Inc, Shanghai, China (Catalogue number: E2298Hu)); Human Calreticulin (CRT) ELISA Kit from Cusabio Technology Llc., Houston, TX, USA (Catalogue number: CSB-E09787h)).

Techniques: