Journal: Communications Biology

Article Title: Cooperation between oncogenic Ras and wild-type p53 stimulates STAT non-cell autonomously to promote tumor radioresistance

doi: 10.1038/s42003-021-01898-5

Figure Lengend Snippet: ( a – i ′) Representative images of dissected eye imaginal discs containing ptip −/− or Ras V12 or Ras V12 ptip −/− double mutant clones (GFP) stained with DAPI to detect DNA or anti-phosphorylated H2AV antibodies to detect DNA damage ( a – c’ ) or anti-p53 ( d - e ′) or anti-dacapo (dap/p21) ( f – g ′) or anti-phosphorylated JNK ( h - i ′) antibodies to detect cellular response to DNA damage. Scale bars are 20 µm. ( j , k ) Quantitative Polymerase Chain Reaction (qPCR) data showing expression of p53 or dap/ p21 in wild type versus ptip −/− eye imaginal discs ( j ) or the expression of p53 in Ras V12 or ptip −/− or Ras V12 ptip −/− eye imaginal discs ( k ). Expression was normalized to the transcript abundance of the housekeeping gene rp49 . Error bars denote standard deviation (SD) values. P values are derived from Student’s t test analyses. ( l – r) Matched light and fluorescence images of adult eyes containing GFP-labeled clones. The respective clone genotypes are indicated at the top of each panel. The corresponding fluorescent images are shown below in ( l ′– r ′). GPF-negative tissues represent wild-type tissues. Scale bars are 150 µm. ( s ) Quantification of the nonautonomous growth phenotype of adult eyes containing clones of the indicated genotypes: Ras V12 ptip −/− , Ras V12 ptip −/− Bsk DN , Ras V12 ptip −/− p53 R155H , or Ras V12 ptip −/− p53 RNAi . ( t – u″ ) Genetic juxtaposition of GFP-labeled Ras V12 clones with RFP-labeled Ras V12 clones ( t - t ″ , controls) or with RFP-labeled clones of cells coexpressing Ras V12 and wild-type p53 ( Ras V12 , p53 OE ) ( u-u ″ ). GFP-positive Ras V12 clones are surrounded by RFP/GFP double-positive Ras V12 clones ( t-t ″ ) or by RFP/GFP double-positive Ras V12 , p53 OE clones ( u-u ″ ). Brain cephalic complex images showing the growth of Ras V12 clones when juxtaposed to Ras V12 or to Ras V12 , p53 OE clones are shown in t and u , respectively. Dotted white lines ( t ′ , t ″ , u ′ , u ″ ) represent tissue boundaries. Scale bars are 100 µm. ( v ) Quantification of eye tissue sizes from ( t – u ″ ). Sample size N = 10 tissues per genotype. Error bars denote standard error of the mean (SEM) values. P values are derived from Student’s t test analyses. Effect size (Cohen’s d values) for ( j ), ( k ), and ( v ) is greater than 0.8.

Article Snippet: The following antibodies were used: mouse monoclonal anti-β gal (1:500, Sigma), mouse anti-dmp53 (1:50 DSHB), rabbit anti-Stat92E (1:1000; gift from S. Hou), rabbit anti p-JNK (1:100), mouse anti-p21 (1:200, DSHB) and mouse anti-H2Av monoclonal antibody (1:200, DSHB #UNC93-5.2.1).

Techniques: Mutagenesis, Clone Assay, Staining, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Derivative Assay, Fluorescence, Labeling

Journal: Communications Biology

Article Title: Cooperation between oncogenic Ras and wild-type p53 stimulates STAT non-cell autonomously to promote tumor radioresistance

doi: 10.1038/s42003-021-01898-5

Figure Lengend Snippet: ( a – d ′) Upregulated p53 and dap/p21 within Ras V12 clones after irradiation. GFP-labelled Ras V12 clones were stained with anti-p53 antibody ( a , a ′, c , c ′) or anti-p21 antibody ( b , b ′, d , d ′) before irradiation (0 h) and 24 h after irradiation. Time was counted from the start of first faction of IR treatment. Scale bar is 20 µm. ( e , f ) Quantification by qPCR of upd , upd2 , and upd3 expression in eye-antennal discs containing wild-type or Ras V12 clones after 36 h of first fraction of IR treatment (IR+) or without IR treatment (IR−). Column bars represent the mean of fold changes for the expression level of indicated genes ( e ). Relative expression of upd2 and upd3 in irradiated eye-antennal discs containing wild type, Ras V12 and Ras V12 p53 R155H clones ( f ). Three independent experiments were carried out. Error bars denote SD. P values are derived from Student’s t test analyses. ( g ) Diagram of setting Drosophila irradiation models. Larvae after egg laying (48 h) were irradiated with three fractions of 10 Gy and allowed to recover to late third-instar larval stage. All eye-antennal discs were dissected at the late third-instar larval stage to evaluate the irradiation results by measuring the relative size between GFP-labeled clones and whole eye-antennal discs. ( h – l ′) GFP-labeled clones homozygous for Ras V12 ( i , i ′), sav 3 ( j , j ′), Tsc1 Q600X ( k , k ′), or expressing dMyc ( l , l ′) as well as wild-type controls ( h , h ′) were induced in the eye-antennal discs of larvae, irradiated at 48 h, and then collected discs on day 5. ( h – l ) the eye-antennal discs without irradiation treatment (IR−). ( h ′– l ′) show irradiated discs (IR+). ( m ) Quantification of relative eye disc size (blue) and GFP-clone size (green) treated with IR (IR+) or without IR (IR−). For each genotype, eye-antennal disc and GFP-clone were normalized to age-matched eye discs without IR. Column bars represent the mean size of samples ( N = 5–10). Scale bar is 50 µm. ( n – t ′) GFP-labeled Ras V12 , p53 −/− , Ras V12 p53 −/− , upd RNAi upd2 ∆ , Ras V12 Upd RNAi upd2 ∆ , Dome DN , and Ras V12 Dome DN clones were induced in the eye-antennal discs and half were then irradiated at the second-instar larval stage. After 3 days of recovery, all eye discs at the late third-instar larval stage were dissected to evaluate the differences in response to irradiation. ( n – t ) Eye-antennal discs without irradiation treatment (IR−) and ( n ′– t ′) eye discs treated with irradiation (IR+). Scale bar is 50 µm. ( u ) Quantification of clones and eye discs treated with or without irradiation. Eye-antennal disc and GFP-clone areas were measured by ImageJ and normalized to the eye-antennal discs with the same genotype at the same age without IR. Column bars represent the mean size of samples ( N = 5–9). Blue columns represent the mean size of the entire eye-antennal tissue for the indicated genotypes; green columns represent the size of GFP-labeled tumors. Error bars denote SEM. P values are derived from Student’s t test analyses. Effect size ( d ) values for e , f , m, and u are greater than 0.8.

Article Snippet: The following antibodies were used: mouse monoclonal anti-β gal (1:500, Sigma), mouse anti-dmp53 (1:50 DSHB), rabbit anti-Stat92E (1:1000; gift from S. Hou), rabbit anti p-JNK (1:100), mouse anti-p21 (1:200, DSHB) and mouse anti-H2Av monoclonal antibody (1:200, DSHB #UNC93-5.2.1).

Techniques: Clone Assay, Irradiation, Staining, Expressing, Derivative Assay, Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: Butorphanol reduces the neuronal inflammatory response and apoptosis via inhibition of p38/JNK/ATF2/p53 signaling

doi: 10.3892/etm.2022.11151

Figure Lengend Snippet: Effect of butorphanol on LPS-induced decrease in PC12 cell viability and p38/JNK/ATF2/p53 signaling. (A) Chemical structure of butorphanol. (B) The effect of different doses of butorphanol on PC12 cell viability was assessed using a CCK8 assay. (C) The viability of cells treated with butorphanol and LPS was determined using a CCK8 assay. (D) The expression levels of p-p38, p-JNK, p-ATF2, p53, p38, JNK and ATF2 were measured using western blot analysis. *** P<0.001 vs. Control group. # P<0.05, ## P<0.01 and ### P<0.001 vs. LPS group. LPS, lipopolysaccharide; p-, phosphorylated; ATF2, activating transcription factor 2.

Article Snippet: Phosphorylated (p-)p38 (cat. no. ab4822; dilution, 1:1,000), p38 (cat. no. ab170099; dilution, 1:2,000), p-JNK (cat. no. ab124956; dilution, 1:5,000), JNK (cat. no. ab208035; dilution, 1:500), ATF2 (cat. no. ab239361; dilution, 1:1,000) and Histone H3 (cat. no. ab1791; dilution, 1:2,000) primary antibodies were obtained from Abcam. p-ATF2 (cat. no. MA5-33115; dilution, 1:1,000), p53 (cat. no. 21891-1-AP; dilution, 1:1,000), p65 (cat. no. 14-6731-81; dilution, 1:1,000), Bcl2 (cat. no. PA5-27094; dilution, 1:1,000), Bax (cat. no. PA5-11378; dilution, 1:2,000), cleaved caspase3 (cat. no. PA5-114687; dilution, 1:1,000), GAPDH (cat. no. MA1-16757; dilution, 1:2,000) and anti-rabbit secondary antibodies (cat. no. G-21234; dilution, 1:100,000) were purchased from Thermo Fisher Scientific.

Techniques: CCK-8 Assay, Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Butorphanol reduces the neuronal inflammatory response and apoptosis via inhibition of p38/JNK/ATF2/p53 signaling

doi: 10.3892/etm.2022.11151

Figure Lengend Snippet: Butorphanol protects cells against the inhibitory effects of LPS on viability via p38/JNK/ATF2/p53 signaling. (A) The protein expression levels of p-p38, p-JNK, p-ATF2, p53, p38, JNK and ATF2 in each group were determined using western blot analysis. (B) Cell viability in each group was assessed using a CCK8 assay. (C) The activity of LDH in each group was measured using assay kits. ** P<0.01 and *** P<0.001 vs. Control group. ## P<0.01 and ### P<0.001 vs. LPS group. ▲ P <0.05, ▲▲ P<0.01 and ▲▲▲ P<0.001 vs. LPS + Butorphanol group. p-, phosphorylated; ATF2, activating transcription factor 2; LDH, lactate dehydrogenase.

Article Snippet: Phosphorylated (p-)p38 (cat. no. ab4822; dilution, 1:1,000), p38 (cat. no. ab170099; dilution, 1:2,000), p-JNK (cat. no. ab124956; dilution, 1:5,000), JNK (cat. no. ab208035; dilution, 1:500), ATF2 (cat. no. ab239361; dilution, 1:1,000) and Histone H3 (cat. no. ab1791; dilution, 1:2,000) primary antibodies were obtained from Abcam. p-ATF2 (cat. no. MA5-33115; dilution, 1:1,000), p53 (cat. no. 21891-1-AP; dilution, 1:1,000), p65 (cat. no. 14-6731-81; dilution, 1:1,000), Bcl2 (cat. no. PA5-27094; dilution, 1:1,000), Bax (cat. no. PA5-11378; dilution, 1:2,000), cleaved caspase3 (cat. no. PA5-114687; dilution, 1:1,000), GAPDH (cat. no. MA1-16757; dilution, 1:2,000) and anti-rabbit secondary antibodies (cat. no. G-21234; dilution, 1:100,000) were purchased from Thermo Fisher Scientific.

Techniques: Expressing, Western Blot, CCK-8 Assay, Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Butorphanol reduces the neuronal inflammatory response and apoptosis via inhibition of p38/JNK/ATF2/p53 signaling

doi: 10.3892/etm.2022.11151

Figure Lengend Snippet: Butorphanol decreases LPS-induced inflammatory factor release via p38/JNK/ATF2/p53 signaling. (A-C) The expression levels of TNF-α, IL-1β and IL-6 in the above four groups were measured using reverse transcription-quantitative PCR. (D) The expression levels of p65 (cytoplasm) and p65 (nucleus) were determined using western blot analysis. *** P<0.001 vs. Control group. ### P<0.001 vs. LPS group. ▲ P <0.05 and ▲▲▲ P<0.001 vs. LPS + Butorphanol group. LPS, lipopolysaccharide.

Article Snippet: Phosphorylated (p-)p38 (cat. no. ab4822; dilution, 1:1,000), p38 (cat. no. ab170099; dilution, 1:2,000), p-JNK (cat. no. ab124956; dilution, 1:5,000), JNK (cat. no. ab208035; dilution, 1:500), ATF2 (cat. no. ab239361; dilution, 1:1,000) and Histone H3 (cat. no. ab1791; dilution, 1:2,000) primary antibodies were obtained from Abcam. p-ATF2 (cat. no. MA5-33115; dilution, 1:1,000), p53 (cat. no. 21891-1-AP; dilution, 1:1,000), p65 (cat. no. 14-6731-81; dilution, 1:1,000), Bcl2 (cat. no. PA5-27094; dilution, 1:1,000), Bax (cat. no. PA5-11378; dilution, 1:2,000), cleaved caspase3 (cat. no. PA5-114687; dilution, 1:1,000), GAPDH (cat. no. MA1-16757; dilution, 1:2,000) and anti-rabbit secondary antibodies (cat. no. G-21234; dilution, 1:100,000) were purchased from Thermo Fisher Scientific.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Butorphanol reduces the neuronal inflammatory response and apoptosis via inhibition of p38/JNK/ATF2/p53 signaling

doi: 10.3892/etm.2022.11151

Figure Lengend Snippet: Butorphanol decreases LPS-induced apoptosis via p38/JNK/ATF2/p53 signaling. (A) Anisomycin increased the number of apoptotic cells. (B) TUNEL assay was used to evaluate cell apoptosis (magnification, x200). (C) Expression levels of apoptosis-related protein were determined using western blot analysis. ** P<0.01 and *** P<0.001 vs. Control group. # P<0.05 and ### P<0.001 vs. LPS group. ▲ P <0.05, ▲▲ P<0.01 and ▲▲▲ P<0.001 vs. LPS + Butorphanol group. LPS, lipopolysaccharide.

Article Snippet: Phosphorylated (p-)p38 (cat. no. ab4822; dilution, 1:1,000), p38 (cat. no. ab170099; dilution, 1:2,000), p-JNK (cat. no. ab124956; dilution, 1:5,000), JNK (cat. no. ab208035; dilution, 1:500), ATF2 (cat. no. ab239361; dilution, 1:1,000) and Histone H3 (cat. no. ab1791; dilution, 1:2,000) primary antibodies were obtained from Abcam. p-ATF2 (cat. no. MA5-33115; dilution, 1:1,000), p53 (cat. no. 21891-1-AP; dilution, 1:1,000), p65 (cat. no. 14-6731-81; dilution, 1:1,000), Bcl2 (cat. no. PA5-27094; dilution, 1:1,000), Bax (cat. no. PA5-11378; dilution, 1:2,000), cleaved caspase3 (cat. no. PA5-114687; dilution, 1:1,000), GAPDH (cat. no. MA1-16757; dilution, 1:2,000) and anti-rabbit secondary antibodies (cat. no. G-21234; dilution, 1:100,000) were purchased from Thermo Fisher Scientific.

Techniques: TUNEL Assay, Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Butorphanol reduces the neuronal inflammatory response and apoptosis via inhibition of p38/JNK/ATF2/p53 signaling

doi: 10.3892/etm.2022.11151

Figure Lengend Snippet: p38/JNK/ATF2/p53 signal pathway diagram. MKK, mitogen-activated protein kinase kinase; + P, phosphorylation; ATF2, activating transcription factor 2.

Article Snippet: Phosphorylated (p-)p38 (cat. no. ab4822; dilution, 1:1,000), p38 (cat. no. ab170099; dilution, 1:2,000), p-JNK (cat. no. ab124956; dilution, 1:5,000), JNK (cat. no. ab208035; dilution, 1:500), ATF2 (cat. no. ab239361; dilution, 1:1,000) and Histone H3 (cat. no. ab1791; dilution, 1:2,000) primary antibodies were obtained from Abcam. p-ATF2 (cat. no. MA5-33115; dilution, 1:1,000), p53 (cat. no. 21891-1-AP; dilution, 1:1,000), p65 (cat. no. 14-6731-81; dilution, 1:1,000), Bcl2 (cat. no. PA5-27094; dilution, 1:1,000), Bax (cat. no. PA5-11378; dilution, 1:2,000), cleaved caspase3 (cat. no. PA5-114687; dilution, 1:1,000), GAPDH (cat. no. MA1-16757; dilution, 1:2,000) and anti-rabbit secondary antibodies (cat. no. G-21234; dilution, 1:100,000) were purchased from Thermo Fisher Scientific.

Techniques:

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 1. Upregulation of polyribonucleotide nucleotidyltransferase 1 (PNPT1) in host cells infected by various viruses and depletion of PNPT1 in host cells markedly reduced viral replication. Immunostaining (a) and Western blot analysis (b) of PNPT1 in BEAS-2B cells with or without adenovirus (Adv-5) infection (24 h). Immunostaining (c) and Western blot analysis (d) of PNPT1 in NIH-3T3 cells with or without murine cytomegalovirus (MCMV) infection (48 h). Immunostaining (e) and Western blot analysis (f) of PNPT1 in Neuro-2a cells with or without murine hepatovirus (MHV-A59) infection (12 h). (g) Quantitative reverse transcription polymerase chain reaction analysis of viral DNA level in BEAS-2B cells (left), NIH-3T3 cells (middle) and Neuro-2a cells (right) with or without Adv-5, MCMV or MHV-A59 infection for 12 h, 24 h or 48 h at indicated multiplicity of infection, respectively. Cells were treated with control shRNA (sh-CTL) or PNPT1 shRNA (sh-PNPT1) prior to viral infection. Scale bar 20 μm. Data from three independent experiments are presented as mean ± standard deviation. ∗∗∗∗P < 0.0 0 01.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Infection, Immunostaining, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Control, shRNA, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 2. A reduction in polyribonucleotide nucleotidyltransferase 1 (PNPT1) promotes relocation of mitochondria double-stranded RNAs (mt-dsRNAs) from mitochondria to cytoplasm. (a–c) Immunostaining of mt-dsRNAs (J2) in BEAS-2B cells infected or not infected with adenovirus (Adv-5) at 24 h (a), NIH-3T3 cells infected or not infected with murine cytomegalovirus (MCMV) at 48 h (b), and Neuro-2a cells infected or not infected with murine hepatovirus (MHV-A59) at 12 h (c). Scale bar 20 μm. (d) Quantitative reverse transcription polymerase chain reaction analysis of cytosolic mt-dsRNAs in BEAS-2B cells infected or not infected with Adv-5 at 24 h. (e) In-situ staining of cytosolic mt- ND5 heavy (left) and light strands (right) in BEAS-2B cells infected or not infected with Adv-5 at 24 h. ATP5A1 served as a mitochondria marker. Scale bar 50 μm. Data from three independent experiments are presented as mean ± standard deviation.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Immunostaining, Infection, Reverse Transcription, Polymerase Chain Reaction, In Situ, Staining, Marker, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 3. Cytosolic mitochondria double-stranded RNAs activate RNA-activated protein kinase (PKR) and, subsequently, eukaryotic initiation factor 2 α (eIF2 α) to terminate general protein production. (a) Left: Fluorescence microscopy images of BEAS-2B cells infected with adenovirus (Adv-5) for 1 h, then washed with phosphate buffered saline; nascent protein synthesis at 24 h post-infection was detected by OPP-647 (red) and DAPI (blue) staining. Right: Quantification of OPP-647 staining. Scale bar, 75 μm. (b) Western blot analysis of PNPT1, phosphorylated PKR (p-PKR), PKR, phosphorylated eIF2 α and eIF2 α levels in BEAS-2B cells infected or not infected with Adv-5 after 24 h infection. (c) Western blot analysis of PNPT1, p-PKR, PKR, p-eIF2 α and eIF2 α levels in NIH/3T3 cells infected or not infected with murine cytomegalovirus (MCMV) after 48 h infection. (d) Western blot analysis of PNPT1, p-PKR, PKR, p-eIF2 α and eIF2 α levels in Neuro-2a cells infected or not infected with murine hepatovirus (MHV-A59) after 12 h infection. In (a–d), cells were treated with control shRNA (sh-CTL) or PNPT1 shRNA (sh-PNPT1) prior to viral infection.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Fluorescence, Microscopy, Infection, Saline, Staining, Western Blot, Control, shRNA

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 4. Identification of lanatoside C (LanC) as a potent inhibitor of polyribonucleotide nucleotidyltransferase 1 (PNPT1) using bioactive compounds approved by the US Food and Drug Administration. (a) Prediction of PNPT1 inhibition based on functional PNPT1 structural analysis. (b) BEAS-2B cells were infected with adenovirus (Adv-5) at multiplicity of infection (MOI) of 0.5 and treated with various compounds at indicated concentrations for 24 h. The effects of each compound on induction of cellular mitochondria double-stranded RNAs (mt-dsRNAs) labelled with J2 antibody were analysed by immunofluorescence staining. (c) Left: Representative images of cellular mt- dsRNA staining by J2 antibody as a readout for PNPT1 inhibition (red, mt-dsRNA; blue, nuclei). Scale bar 50 μm. Right: Quantification of cellular mt-dsRNAs labelled with J2 antibody was analysed by immunofluorescence staining. (d) BEAS-2B cells were infected with Adv-5 at MOI of 0.5 and treated with different com pounds at indicated concentrations for 24 h. The inhibition of viral replication was analysed using quantitative reverse transcription polymerase chain reaction. (e) Dose-dependent inhibition of LanC and atazanavir sulfate on Adv-5 replication (MOI of 0.5) in BEAS-2B cells. (f) Micro-scale thermophoresis analysis of binding affinity of LanC with functional PNPT1 trimer. Data from three independent experiments are presented as mean ± standard deviation. ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Inhibition, Functional Assay, Infection, Staining, Reverse Transcription, Polymerase Chain Reaction, Binding Assay, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 6. Lanatoside C (LanC), an inhibitor of polyribonucleotide nucleotidyltransferase 1 (PNPT1), suppresses virus replication by activating RNA-activated protein kinase (PKR) and, subsequently, eukaryotic initiation factor 2 α (eIF2 α). (a) Immunostaining of double-stranded RNA (red) in BEAS-2B cells treated with LanC at indicated concentration for 24 h. Scale bar 20 μm. (b) Quantitative reverse transcription polymerase chain reaction analysis of cytosolic mitochondria double-stranded RNAs (mt-dsRNAs) in BEAS- 2B cells infected and not infected with adenovirus (Adv-5). (c) In-situ staining of cytosolic mt- ND5 heavy (left) and light strands (right) in BEAS-2B cells infected and not infected with Adv-5. ATP5A1 served as a mitochondria marker. Scale bar 50 μm. (d) Western blot analysis of PNPT1, p-PKR, PKR, p-eIF2 and eIF2 expression in BEAS-2B cells treated with LanC at indicated concentrations for 24 h. (e) Left: Fluorescence microscopy images of BEAS-2B cells infected with Adv-5 virus for 1 h, washed with phosphate buffered saline, then treated with LanC at indicated concentrations for another 24 h; nascent protein synthesis was detected by staining with OPP-647 (red), and genomic DNA was stained using DAPI (blue). Right: Quantification of OPP-647 staining. Scale bar 50 μm. Data from three independent experiments are presented as mean ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0 0 01.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Virus, Immunostaining, Concentration Assay, Reverse Transcription, Polymerase Chain Reaction, Infection, In Situ, Staining, Marker, Western Blot, Expressing, Fluorescence, Microscopy, Saline, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 7. Lanatoside C (LanC) suppresses production of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike (S) protein in BEAS-2B cells. BEAS-2B cells were infected with SARS-CoV-2 pseudo-virions at multiplicity of infection of 0.01, and treated with LanC at indicated concentrations for 24 h. (a) The effects of suppression of viral replication were analysed quantitatively using quantitative reverse transcription polymerase chain reaction. (b) The effects of suppression of viral replication were detected with green fluorescent protein (GFP). (c) Western blot analysis of S-protein, polyribonucleotide nucleotidyltransferase 1 (PNPT1), phosphorylated RNA-activated protein kinase (p-PKR), PKR, phosphorylated eukaryotic initiation factor 2 α (p-eIF2 α) and eIF2 α expression after LanC treatment. Data from three independent experiments are presented as mean ± standard deviation. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0 0 01.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Infection, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Expressing, Standard Deviation

Journal: International journal of antimicrobial agents

Article Title: Blockade of pan-viral propagation by inhibition of host cell PNPT1.

doi: 10.1016/j.ijantimicag.2024.107124

Figure Lengend Snippet: Fig. 8. Schematic working model of lanatoside C (LanC), an inhibitor of polyribonucleotide nucleotidyltransferase 1 (PNPT1), blocking pan-viral replication in host cells. PKR, RNA-activated protein kinase; eIF2 α, eukaryotic initiation factor 2 α.

Article Snippet: Cells ere blocked in 3% (w/v) bovine serum antigen diluted in phoshate buffered saline (PBS) solution for 30 min, and then inubated overnight with the monoclonal anti-dsRNA J2 antibody 10010200; Scicons, Susteren, The Netherlands) and PNPT1 polylonal antibody (14487-1-AP; Proteintech), followed by incubation ith fluorophore-tagged secondary antibodies (Thermo Fisher).

Techniques: Blocking Assay

Journal: Clinical science (London, England : 1979)

Article Title: Inhibition of HDAC3 prevents diabetic cardiomyopathy in OVE26 mice via epigenetic regulation of DUSP5-ERK1/2 pathway

doi: 10.1042/CS20170064

Figure Lengend Snippet: Representative Western blot of whole cell p-ERK1/2 and ERK1/2, nuclear p-ERK1/2 and ERK1/2 (A, C), and densitometric analyses of Western blot of p-ERK1/2 protein levels (B, D). The expression of DUSP5 detected by real-time PCR (E) and Western blot (F, G). The acetylated level of histone H3 on the DUSP5 gene promoter region revealed by ChIP (H). Normal mouse IgG was used as a negative control and data were normalized to input DNA samples. Data are presented as the mean ± S.D. n=5–7. *P < 0.05 vs. WT-Veh; #P < 0.05 vs. OVE-Veh at the same time point. Schematic illustration of the working hypothesis for the regulation of nuclear ERK1/2 signaling and diabetes-induced cardiac injury by HDAC3 and DUSP5 (I). Diabetes increased ERK1/2 phosphorylation and nuclear accumulation of phosphorylated ERK1/2 where it turns on the cardiac apoptosis, hypertrophy and fibrosis signaling pathways, leading to the final cardiac remodeling and dysfunction. HDAC3 (or HDACs) inhibitor stimulates DUSP5 expression by increasing the acetylated histone H3, leading to the dephosphorylation of nuclear ERK1/2 and thus reducing cardiac remodeling and dysfunction. In addition, phosphorylated ERK1/2 also inhibits DUSP5 expression via stimulating HDACs, making a self-reinforcing cycle.

Article Snippet: Primers [ANP: Mm01256744_m1; collagen I: Mm01302043_g1; fibronectin-1 (FN-1): Mm01255747_g1; DUSP5: Mm01266106; GAPDH: Mm99999915_g1] for PCR were obtained from Thermo Fisher (Grand Island, NY, USA).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Negative Control, Phospho-proteomics, Protein-Protein interactions, De-Phosphorylation Assay