Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a Levels of the cytokines in cell culture supernatants from S41- or C2-infected neuronal cultures collected over a 96 h infection course. Uninfected neuronal culture was used as control. CXCL1 showed the highest increase during HEVA71 infection. G-CSF exhibited a delay in its upregulation in contrast to MCP-1, MIP1α, RANTES and CXCL1. Dotted lines represent the peak cytokine concentration that mostly corresponded to peak viral titers detected at 24 hpi for DIV7 neuronal cultures. b Immunostaining of S41-, C2- or UI-infected astrocytes with antibodies against dsRNA and GFAP. Scale bars represent 20 µm. Strong dsRNA-positive signals were detected in HEVA71-infected astrocytes (arrowheads) but not in UI controls. c Quantification of dsRNA-positive cells in infected astrocytes at 24 hpi. Two hundred cells taken from random fields were manually counted for each condition. d CXCL1 was elevated in cell culture supernatants of HEVA71-infected astrocyte cultures but not in UI controls at 24 hpi. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (**** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Cell Culture, Infection, Concentration Assay, Immunostaining

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a Survival rates and b mean clinical scores of mice infected with S41 or UI. S41-infected mice but not UI-infected mice displayed mortality and high clinical scores from 4 dpi. c High viral loads were detected in brains of S41- but not UI-infected mice (below LOD = 0.2) at 7 dpi. d , e CXCL1 levels were slightly elevated in d serum but highly elevated in e CSF of S41-infected mice but not in UI-infected mice that correlated with neuropathological deficits seen only in S41-infected mice. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3, 10 mice were used for each group per independent experiment). f CSF CXCL1 chemokine concentrations in severe HEVA71 HFMD patients significantly exceeds CXCL1 levels of other groups. Violin plots represent all analyzed values. Bold and dashed lines indicated median and the 25 th and 75 th percentile, respectively. Number of patients per group is indicated on the x -axis. Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001). Exact P values for: moderate HEVA71 HFMD (CSF) = 0.0033, severe CA6 HFMD (CSF) = 0.0301, severe CA16 HFMD (CSF) = 0.0038 and JEV encephalitis (CSF) = 0.0401. Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Infection

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a HEVA71 viral titers increase in CXCL1-pretreated DIV7 neuronal cultures. Exact P values for: 25 U/ml at 24 hpi = 0.0033 and 48 hpi = 0.0009. b Immunoblotting of cell lysates from CXCL1-pretreated or untreated S41-infected DIV7 neuronal cultures against VP0, VP2, ERK1/2, pERK1/2, JAK2, pS727 (STAT3), pT705 (STAT3) and STAT3. CXCL1 pre-treatment accelerates the appearance HEVA71 viral proteins. Elevated pERK1/2 and ERK1/2 levels also emerged earlier. Immunoblot representative of 3 biological repeats is shown. Bar charts below show the quantifications for each protein. c High levels of VP0 and VP2 were detectable at 12 hpi only with exposure to CXCL1. Exact P values for VP0: 25 U/ml at 24 hpi = 0.0195, 50 U/ml at 24 hpi = 0.0001 and 100 U/ml at 24 hpi = 0.002. Exact P values for VP2: 25 U/ml at 24 hpi = 0.001 and 100 U/ml at 24 hpi = 0.0215. d pERK1/2 to ERK1/2 ratios were higher in pretreated cultures versus controls in a CXCL1 dose-dependent manner at all timepoints. Exact P value for pERK1 to ERK1 ratio: 25 U/ml at 48 hpi = 0.0094. β-actin was used as a loading control for immunoblotting experiments. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Western Blot, Infection

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a Inhibiting ERK1/2 decreased viral titers at 12 and 24 hpi in DIV7 neuronal cultures. b Immunoblotting of S41-infected DIV7 neuronal cultures treated simultaneously with FR180204 (5 µM) and CXCL1 (100 U/ml) at 12, 24 and 48 hpi. ERK1/2 inhibition delays the expression of HEVA71 viral proteins. Immunoblot representative of 3 biological repeats is shown. Bar charts below show the quantifications for each protein. c VP0 and VP2 levels decreased at 12 and 24 hpi with ERK1/2 inhibition. d pERK1/2 to ERK1/2 ratios were reduced at all timepoints in FR180204-treated cultures. Exact P value for pERK1 to ERK1 ratio with FR180204 at 48 hpi = 0.0001. β-actin was used as a loading control for immunoblotting experiments. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments (n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (*** P < 0.001 and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Western Blot, Infection, Inhibition, Expressing

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a CXCR2 deletion in infected neuronal cultures reduced viral titers at all time points tested. b Lysates from neuronal cultures infected with control ( LUC sgRNA)- or CXCR2 sgRNA-expressing lentiviruses were similarly immunoblotted as in a . CXCR2 deletion delays viral protein expression in neuronal cultures despite CXCL1 pretreatment. Immunoblot representative of 3 biological repeats is shown. Bar charts below show the quantifications for each protein. c VP0 and VP2 levels decreased at all timepoints in CXCR2 -deleted samples. d pERK1/2 to ERK1/2 ratios were decreased in CXCR2 -deleted samples. β-actin was used as a loading control for immunoblotting experiments. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (**** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Infection, Expressing, Western Blot

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a CXCL1 levels were elevated in culture supernatants when VP4 and VP4-mut were expressed. Higher levels of the chemokine were detected in samples expressing wildtype VP4. b Immunofluorescence images of control (EGFP)- or VP4 (wildtype and mutant)- expressing neuronal cultures. Neurite disintegration was only observed in wildtype VP4-expressing neuronal cultures. Scale bars represent 20 µm. c Immunoblots of cell lysates from control (EGFP)-, VP4- or VP4-mut-expressing neurons against VP4, EGFP, ERK1/2, pERK1/2, pS727 (STAT3), JAK2, pT705 (STAT3), STAT3, caspase 3 and cleaved caspase 3. Immunoblot representative of 3 biological repeats is shown. Bar charts below show quantifications for each protein. d pERK1/2 to ERK1/2 ratios were higher in VP4-expressing neuronal cultures as compared to VP4-mut or EGFP controls at all time points. e Cleaved caspase 3 was detected only in VP4-expressing neuronal cultures. Exact P value for caspase 3: VP4-mut at 6 dpi = 0.6026. β-actin was used as a loading control for immunoblotting experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (ns > 0.9999 unless specified, and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Mutagenesis, Western Blot

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a – e Infection of mice with wildtype or VP4-mutant HEVA71 virons. a Survival rates and b mean clinical scores of mice infected with wildtype, VP4-mutant or UI virus. Wildtype and VP4-mutant HEVA71-infected mice displayed mortality and high clinical scores from 4 and 7 dpi, respectively. UI-infected mice were healthy till 15 dpi. c VP4-mutant HEVA71-infected mice had lower viral loads detected in their brains at 7 dpi. UI-infected mice were used as controls. Negligible viral loads were detected below limit of detection (LOD = 0.2). d , e CXCL1 levels were highly elevated in d CSF but only slightly elevated in e serum of both wildtype and VP4-mutant HEVA71-infected mice but not in UI-infected mice. Wildtype HEVA71-infected mice showed increasing CXCL1 from 4 dpi but those for VP4-mutant-infected mice emerged at 7 dpi. f Brain lysates of wildtype, VP4-mutant or UI-infected mice were analyzed by immunoblotting at 7 dpi. Infection with VP4-mutant HEVA71 delays the expression of HEVA71 viral proteins. Immunoblot representative of 3 biological repeats is shown. Bar charts below show quantifications for each protein. g VP0 and VP2 levels decreased at 7 dpi with VP4-mutant HEVA71. Exact P values for: VP0 = 0.0009 and VP2 = 0.0002. h pERK1/2 were reduced in VP4-mutant HEVA71-infected mice. Exact P values for: pERK1 = 0.0009 and pERK2 = 0.0012. i Cleaved caspase 3 was detected in wildtype HEVA71-infected mice but not VP4-mutant HEVA71-infected mice at 7 dpi. Exact P values for: caspase 3 = 0.0254 and cleaved caspase 3 = 0.0008. β-actin was used as a loading control for immunoblotting experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (** P < 0.01, *** P < 0.001 and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Infection, Mutagenesis, Western Blot, Expressing

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a Survival rates and b mean clinical scores of mice infected with S41 or UI with or without AZD5069 treatment, respectively. Arrowheads indicate time points of AZD5069 administration. S41-infected mice displayed mortality and high clinical scores from 4 dpi. AZD5069 administration to S41-infected mice delayed mortality and high clinical scores until 8 dpi. UI-infected mice were healthy till 15 dpi. c AZD5069 treatment lowered viral loads in brains of S41-infected mice from 4 to 7 dpi. d , e CXCL1 levels were slightly elevated in d serum but highly elevated in e CSF of S41-infected mice with or without AZD5069 but not in UI-infected mice. f Brain lysates of S41- or UI-infected mice with or without AZD5069 treatment were subjected to immunoblotting analyses at 7 dpi. AZD5069 treatment delays the expression of HEVA71 viral proteins. Immunoblot representative of 3 biological repeats is shown. Bar charts below show quantifications for each protein. g VP0 and VP2 levels decreased at 7 dpi with AZD5069 treatment. Exact P values for: VP0 = 0.0006 and VP2 = 0.0009. h pERK1/2 and ERK1/2 levels were reduced in AZD5069-treated mice. Exact P values for: ERK1 = 0.0002, ERK2 = 0.0013, pERK1 = 0.0001 and pERK2 = 0.0007. i JAK2 levels persisted in CXCR2-suppressed mice with S41 infection. Exact P values for: JAK2 = 0.001. j AZD5069 treatment increased phosphorylation of STAT3 at pT705 but decreased pS727 levels at 7 dpi. Exact P values for: pT705 = 0.0007. k Cleaved caspase 3 was detected only in S41-infected mice without AZD5069 treatment at 7 dpi. Exact P values for: caspase 3 = 0.0013 and cleaved caspase 3 = 0.0015. β-actin was used as a loading control for immunoblotting experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (** P < 0.01, *** P < 0.001 and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Infection, Western Blot, Expressing

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a Levels of the cytokines in cell culture supernatants from S41- or C2-infected neuronal cultures collected over a 96 h infection course. Uninfected neuronal culture was used as control. CXCL1 showed the highest increase during HEVA71 infection. G-CSF exhibited a delay in its upregulation in contrast to MCP-1, MIP1α, RANTES and CXCL1. Dotted lines represent the peak cytokine concentration that mostly corresponded to peak viral titers detected at 24 hpi for DIV7 neuronal cultures. b Immunostaining of S41-, C2- or UI-infected astrocytes with antibodies against dsRNA and GFAP. Scale bars represent 20 µm. Strong dsRNA-positive signals were detected in HEVA71-infected astrocytes (arrowheads) but not in UI controls. c Quantification of dsRNA-positive cells in infected astrocytes at 24 hpi. Two hundred cells taken from random fields were manually counted for each condition. d CXCL1 was elevated in cell culture supernatants of HEVA71-infected astrocyte cultures but not in UI controls at 24 hpi. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (**** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Cell Culture, Infection, Control, Concentration Assay, Immunostaining

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a Survival rates and b mean clinical scores of mice infected with S41 or UI. S41-infected mice but not UI-infected mice displayed mortality and high clinical scores from 4 dpi. c High viral loads were detected in brains of S41- but not UI-infected mice (below LOD = 0.2) at 7 dpi. d , e CXCL1 levels were slightly elevated in d serum but highly elevated in e CSF of S41-infected mice but not in UI-infected mice that correlated with neuropathological deficits seen only in S41-infected mice. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3, 10 mice were used for each group per independent experiment). f CSF CXCL1 chemokine concentrations in severe HEVA71 HFMD patients significantly exceeds CXCL1 levels of other groups. Violin plots represent all analyzed values. Bold and dashed lines indicated median and the 25 th and 75 th percentile, respectively. Number of patients per group is indicated on the x -axis. Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001). Exact P values for: moderate HEVA71 HFMD (CSF) = 0.0033, severe CA6 HFMD (CSF) = 0.0301, severe CA16 HFMD (CSF) = 0.0038 and JEV encephalitis (CSF) = 0.0401. Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Infection

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a HEVA71 viral titers increase in CXCL1-pretreated DIV7 neuronal cultures. Exact P values for: 25 U/ml at 24 hpi = 0.0033 and 48 hpi = 0.0009. b Immunoblotting of cell lysates from CXCL1-pretreated or untreated S41-infected DIV7 neuronal cultures against VP0, VP2, ERK1/2, pERK1/2, JAK2, pS727 (STAT3), pT705 (STAT3) and STAT3. CXCL1 pre-treatment accelerates the appearance HEVA71 viral proteins. Elevated pERK1/2 and ERK1/2 levels also emerged earlier. Immunoblot representative of 3 biological repeats is shown. Bar charts below show the quantifications for each protein. c High levels of VP0 and VP2 were detectable at 12 hpi only with exposure to CXCL1. Exact P values for VP0: 25 U/ml at 24 hpi = 0.0195, 50 U/ml at 24 hpi = 0.0001 and 100 U/ml at 24 hpi = 0.002. Exact P values for VP2: 25 U/ml at 24 hpi = 0.001 and 100 U/ml at 24 hpi = 0.0215. d pERK1/2 to ERK1/2 ratios were higher in pretreated cultures versus controls in a CXCL1 dose-dependent manner at all timepoints. Exact P value for pERK1 to ERK1 ratio: 25 U/ml at 48 hpi = 0.0094. β-actin was used as a loading control for immunoblotting experiments. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Western Blot, Infection, Control

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a Inhibiting ERK1/2 decreased viral titers at 12 and 24 hpi in DIV7 neuronal cultures. b Immunoblotting of S41-infected DIV7 neuronal cultures treated simultaneously with FR180204 (5 µM) and CXCL1 (100 U/ml) at 12, 24 and 48 hpi. ERK1/2 inhibition delays the expression of HEVA71 viral proteins. Immunoblot representative of 3 biological repeats is shown. Bar charts below show the quantifications for each protein. c VP0 and VP2 levels decreased at 12 and 24 hpi with ERK1/2 inhibition. d pERK1/2 to ERK1/2 ratios were reduced at all timepoints in FR180204-treated cultures. Exact P value for pERK1 to ERK1 ratio with FR180204 at 48 hpi = 0.0001. β-actin was used as a loading control for immunoblotting experiments. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments (n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (*** P < 0.001 and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Western Blot, Infection, Inhibition, Expressing, Control

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a CXCR2 deletion in infected neuronal cultures reduced viral titers at all time points tested. b Lysates from neuronal cultures infected with control ( LUC sgRNA)- or CXCR2 sgRNA-expressing lentiviruses were similarly immunoblotted as in a . CXCR2 deletion delays viral protein expression in neuronal cultures despite CXCL1 pretreatment. Immunoblot representative of 3 biological repeats is shown. Bar charts below show the quantifications for each protein. c VP0 and VP2 levels decreased at all timepoints in CXCR2 -deleted samples. d pERK1/2 to ERK1/2 ratios were decreased in CXCR2 -deleted samples. β-actin was used as a loading control for immunoblotting experiments. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (**** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Infection, Control, Expressing, Western Blot

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a CXCL1 levels were elevated in culture supernatants when VP4 and VP4-mut were expressed. Higher levels of the chemokine were detected in samples expressing wildtype VP4. b Immunofluorescence images of control (EGFP)- or VP4 (wildtype and mutant)- expressing neuronal cultures. Neurite disintegration was only observed in wildtype VP4-expressing neuronal cultures. Scale bars represent 20 µm. c Immunoblots of cell lysates from control (EGFP)-, VP4- or VP4-mut-expressing neurons against VP4, EGFP, ERK1/2, pERK1/2, pS727 (STAT3), JAK2, pT705 (STAT3), STAT3, caspase 3 and cleaved caspase 3. Immunoblot representative of 3 biological repeats is shown. Bar charts below show quantifications for each protein. d pERK1/2 to ERK1/2 ratios were higher in VP4-expressing neuronal cultures as compared to VP4-mut or EGFP controls at all time points. e Cleaved caspase 3 was detected only in VP4-expressing neuronal cultures. Exact P value for caspase 3: VP4-mut at 6 dpi = 0.6026. β-actin was used as a loading control for immunoblotting experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (ns > 0.9999 unless specified, and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Mutagenesis, Western Blot

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a – e Infection of mice with wildtype or VP4-mutant HEVA71 virons. a Survival rates and b mean clinical scores of mice infected with wildtype, VP4-mutant or UI virus. Wildtype and VP4-mutant HEVA71-infected mice displayed mortality and high clinical scores from 4 and 7 dpi, respectively. UI-infected mice were healthy till 15 dpi. c VP4-mutant HEVA71-infected mice had lower viral loads detected in their brains at 7 dpi. UI-infected mice were used as controls. Negligible viral loads were detected below limit of detection (LOD = 0.2). d , e CXCL1 levels were highly elevated in d CSF but only slightly elevated in e serum of both wildtype and VP4-mutant HEVA71-infected mice but not in UI-infected mice. Wildtype HEVA71-infected mice showed increasing CXCL1 from 4 dpi but those for VP4-mutant-infected mice emerged at 7 dpi. f Brain lysates of wildtype, VP4-mutant or UI-infected mice were analyzed by immunoblotting at 7 dpi. Infection with VP4-mutant HEVA71 delays the expression of HEVA71 viral proteins. Immunoblot representative of 3 biological repeats is shown. Bar charts below show quantifications for each protein. g VP0 and VP2 levels decreased at 7 dpi with VP4-mutant HEVA71. Exact P values for: VP0 = 0.0009 and VP2 = 0.0002. h pERK1/2 were reduced in VP4-mutant HEVA71-infected mice. Exact P values for: pERK1 = 0.0009 and pERK2 = 0.0012. i Cleaved caspase 3 was detected in wildtype HEVA71-infected mice but not VP4-mutant HEVA71-infected mice at 7 dpi. Exact P values for: caspase 3 = 0.0254 and cleaved caspase 3 = 0.0008. β-actin was used as a loading control for immunoblotting experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (** P < 0.01, *** P < 0.001 and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Infection, Mutagenesis, Virus, Western Blot, Expressing, Control

Journal: Nature Communications

Article Title: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

doi: 10.1038/s41467-022-28533-z

Figure Lengend Snippet: a Survival rates and b mean clinical scores of mice infected with S41 or UI with or without AZD5069 treatment, respectively. Arrowheads indicate time points of AZD5069 administration. S41-infected mice displayed mortality and high clinical scores from 4 dpi. AZD5069 administration to S41-infected mice delayed mortality and high clinical scores until 8 dpi. UI-infected mice were healthy till 15 dpi. c AZD5069 treatment lowered viral loads in brains of S41-infected mice from 4 to 7 dpi. d , e CXCL1 levels were slightly elevated in d serum but highly elevated in e CSF of S41-infected mice with or without AZD5069 but not in UI-infected mice. f Brain lysates of S41- or UI-infected mice with or without AZD5069 treatment were subjected to immunoblotting analyses at 7 dpi. AZD5069 treatment delays the expression of HEVA71 viral proteins. Immunoblot representative of 3 biological repeats is shown. Bar charts below show quantifications for each protein. g VP0 and VP2 levels decreased at 7 dpi with AZD5069 treatment. Exact P values for: VP0 = 0.0006 and VP2 = 0.0009. h pERK1/2 and ERK1/2 levels were reduced in AZD5069-treated mice. Exact P values for: ERK1 = 0.0002, ERK2 = 0.0013, pERK1 = 0.0001 and pERK2 = 0.0007. i JAK2 levels persisted in CXCR2-suppressed mice with S41 infection. Exact P values for: JAK2 = 0.001. j AZD5069 treatment increased phosphorylation of STAT3 at pT705 but decreased pS727 levels at 7 dpi. Exact P values for: pT705 = 0.0007. k Cleaved caspase 3 was detected only in S41-infected mice without AZD5069 treatment at 7 dpi. Exact P values for: caspase 3 = 0.0013 and cleaved caspase 3 = 0.0015. β-actin was used as a loading control for immunoblotting experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments ( n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (** P < 0.01, *** P < 0.001 and **** P < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Culture supernatants from HEVA71-infected neurons, HEVA71-infected astrocytes and lentiviral-infected neurons or CSF and sera taken from mice or human clinical cases over various time/day points were assayed using the respective Bio-Plex Pro rat cytokine 23-plex kit (Bio-Rad) or rat CXCL1 ELISA kit (Aviva Systems Biology) or human CXCL1 ELISA kit (ThermoFisher Scientific) according to manufacturer’s instructions.

Techniques: Infection, Western Blot, Expressing, Phospho-proteomics, Control