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. 2024 Jan;16(1):185-217.
doi: 10.1038/s44321-023-00014-w. Epub 2024 Jan 2.

Methotrimeprazine is a neuroprotective antiviral in JEV infection via adaptive ER stress and autophagy

Surendra K Prajapat  1 , Laxmi Mishra  1 , Sakshi Khera  1 , Shadrack D Owusu  1   2 , Kriti Ahuja  3 , Puja Sharma  1 , Eira Choudhary  1 , Simran Chhabra  1 , Niraj Kumar  4 , Rajan Singh  5   6 , Prem S Kaushal  4 , Dinesh Mahajan  7 , Arup Banerjee  1 , Rajender K Motiani  3 , Sudhanshu Vrati  1 , Manjula Kalia  8
Affiliations

Methotrimeprazine is a neuroprotective antiviral in JEV infection via adaptive ER stress and autophagy

Surendra K Prajapat et al. EMBO Mol Med. 2024 Jan.

Abstract

Japanese encephalitis virus (JEV) pathogenesis is driven by a combination of neuronal death and neuroinflammation. We tested 42 FDA-approved drugs that were shown to induce autophagy for antiviral effects. Four drugs were tested in the JE mouse model based on in vitro protective effects on neuronal cell death, inhibition of viral replication, and anti-inflammatory effects. The antipsychotic phenothiazines Methotrimeprazine (MTP) & Trifluoperazine showed a significant survival benefit with reduced virus titers in the brain, prevention of BBB breach, and inhibition of neuroinflammation. Both drugs were potent mTOR-independent autophagy flux inducers. MTP inhibited SERCA channel functioning, and induced an adaptive ER stress response in diverse cell types. Pharmacological rescue of ER stress blocked autophagy and antiviral effect. MTP did not alter translation of viral RNA, but exerted autophagy-dependent antiviral effect by inhibiting JEV replication complexes. Drug-induced autophagy resulted in reduced NLRP3 protein levels, and attenuation of inflammatory cytokine/chemokine release from infected microglial cells. Our study suggests that MTP exerts a combined antiviral and anti-inflammatory effect in JEV infection, and has therapeutic potential for JE treatment.

Keywords: Antipsychotic; Microglia; Neuroinflammation; Phenothiazines; Trifluoperazine.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1. Antiviral and anti-inflammatory effect of FDA-approved drugs against JEV infection.
(A,B) Neuro2a cells were infected with JEV (MOI 1), and at 1 hpi treated with DMSO/drugs (10 µM). (A) Cells were harvested at 24 hpi and viral RNA levels were quantified using qRT-PCR. Graph shows the relative expression levels of JEV RNA normalized to DMSO-treated control. Data is plotted from two independent experiments (n = 6). (B) Culture supernatant was collected to determine extracellular virus titers using plaque assays. Data represents values obtained from two independent experiments (n = 4). (C–F) N9 cells were mock/JEV (MOI 1) infected, and at 12 hpi treated with DMSO/drugs (10 µM) for 24 h. Cytokine concentrations (pg/ml) were quantified from the culture supernatant using CBA assay. Data shows values from one representative experiment with biological triplicates (n = 3). (G,H) Neuro2a cells were mock/JEV (MOI 5) infected, and at 1 hpi were treated with DMSO/drugs (10 µM) till 24 hpi. (G) Cells were immunostained for JEV NS1 (green) and images were acquired on high-content imaging system. Scale bar, 10 µm. (H) Bar-graph showing percentage of NS1 positive cells from two independent experiments (n = 6). (I) Neuro2a cells were mock/JEV (MOI 5) infected, and at 1 hpi were treated with DMSO/FDA-drugs (10 µM); or at 16 hpi treated with DMF (70 µM)/NAC (3 mM), and maintained till 24 hpi. Post-treatment, cells were stained with oxidative stress indicator CM-H2DCFDA and fluorescence intensity was measured using flow cytometry. The graph represents mean fluorescence intensity values (n = 3). Data information: All data are expressed as means ± SD, statistical significance was determined using one-way ANOVA followed by Dunnett test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.
Figure 2
Figure 2. Efficacy of MTP in JEV-mouse model.
Three-week-old C57BL/6 mice were mock/JEV-S3 (107 pfu) infected through an i.p. injection, and at 4 hpi were treated with vehicle control (PEG400) or MTP (2 mg/kg) by oral gavage at an interval of 24 h for 15 days. All mice were monitored for the appearance of encephalitis symptoms until death. (A,B) (A) Survival curve of mock (n = 4)/MTP (n = 4)/JEV (n = 6)/JEV + MTP (n = 6), Log-rank (Mantel-Cox) test was used to determine the statistical significance comparing JEV and JEV + MTP mice group. (B) Graph representing the change in body weight of vehicle/MTP-treated infected mice group normalized to mock-infected mice group, compared by unpaired Student t-test. (C) Mock or Vehicle/MTP treated infected mice (n = 3/time point from each group) were sacrificed at indicated time points. Brain tissues were homogenized and the supernatant was used for JEV titration using plaque assay. Each data point denotes one mouse, and the virus titers between JEV and JEV + MTP group was compared by unpaired Student t-test. (D,E) Mock or Vehicle/MTP treated infected mice received an i.p. injection of 2% Evans blue dye (100 µl). Mice were sacrificed at 3 and 6 dpi. (D) Representative images showing Evans blue dye distribution in the brain (n = 3). (E) The brain tissues were homogenized in DMF (200 mg/500 μl DMF) and absorbance was measured at 620 nm. The concentration of Evans blue was quantitated according to standard curve and significance was compared by two-way ANOVA followed by Tukey test. (F) Brain tissue was collected at indicated time points, and an equal amount of protein (30 µg) from each sample was used for the quantitation of cytokine levels using CBA. Data were analyzed with LEGENDplexTM Multiplex assay software, and significance was compared by one-way ANOVA followed by Dunnett test (n = 3/time point from each group). Data information: All data expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.
Figure 3
Figure 3. Phenothiazines exert antiviral and anti-inflammatory effects.
(A) Chemical structure of the phenothiazine ring and its derivatives Methotrimeprazine (MTP) and Trifluoperazine (TFP). (B–E) Neuro2a cells (B,C), and cortical neurons (D,E), were mock/JEV (MOI 1) infected, and at 1 hpi were treated with DMSO/MTP at indicated concentrations till 24 hpi. Cells were harvested, qRT-PCR was performed for the quantitation of JEV RNA levels, and % cell viability was measured using MTT assay. Data were normalized to DMSO control (n ≥ 3), and compared between DMSO and MTP treated JEV-infected cells. Graphs represent the CC50 and IC50 values (B,D). Virus titers in the culture supernatants of Neuro2a cells (C), cortical neurons (E) was determined by plaque assays (n = 3). Statistical significance was determined using one-way ANOVA followed by Dunnett test. (F,G) Neuro2a cells were mock/JEV (MOI 1) infected and treated with DMSO/TFP at the indicated concentrations till 24 hpi. CC50 and IC50 were calculated as described above. (G) Culture supernatant was collected and virus titers was determined using plaque assay (n = 3). Statistical significance was determined using one-way ANOVA followed by Dunnett test. (H,I) Neuro2a cells were mock/JEV (MOI 5) infected, and at 1 hpi treated with 10 µM TFP till 24 hpi. Cells were immunostained with JEV NS1 antibody, and images were visualized by high-content imaging system, representative images are shown. Scale bar, 10 µm (H). (I) Graph shows % NS1 positive cells (n = 3), unpaired Student t-test. (J) N9 cells were infected with JEV (MOI 1), at 12 hpi cells were treated with TFP (10 µM) for 24 h. Cytokines were quantified from the soup using CBA (n = 3), unpaired Student t-test. (K) C57BL/6 (3 weeks old) mice were infected by i.p. injection of DMEM (mock) or JEV (106 pfu), and at 4 hpi treated with vehicle control (PEG400)/TFP (1 mg/kg) by oral route at an interval of 24 h till 15 days. All mice were monitored for JEV symptoms until death. The survival curve of mock (n = 4)/TFP (n = 4)/JEV (n = 19)/JEV + TFP (n = 19) was plotted from two independent experiments. Log-rank (Mantel-Cox) test was used to determine the statistical significance comparing vehicle and drug-treated infected mice group. Data information: All data expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.
Figure 4
Figure 4. Phenothiazines are mTOR-independent autophagy inducers.
(A–D) Neuro2a (A,B), and cortical neurons (C,D), were treated with DMSO/Torin1 (1 µM)/MTP (10 µM)/TFP (10 µM) for 6 h. Protein lysates were immunoblotted for LC3 and GAPDH (loading control). (B,D) Bar-graph shows relative expression of LC3II/GAPDH normalized to DMSO control from three independent experiments. (E,F) Neuro2a (E), and cortical neurons (F), were treated with DMSO/Torin1 (1 µM)/MTP (10 µM)/TFP (10 µM) for 4 h, followed by Baf-A1 (100 nM) treatment for 2 h. The values below the blot show relative levels of LC3II/GAPDH protein after normalization to DMSO-treated cells. (G,H) Cortical neurons were treated as described in panel (A), protein lysates were immunoblotted for P62 and GAPDH (loading control). (H) Bar-graph shows relative expression of P62/GAPDH normalized to DMSO control from three independent experiments. (I–O) Neuro2a cells were treated with DMSO/Torin1 (1 µM)/MTP (10 µM)/TFP (10 µM) for 6 h, protein lysates were analyzed by western blotting with P62 (I,J), p-mTOR (S2448), mTOR (I–K), p-P70S6K (Thr386), p70S6K (L,M), p-4E-BP (Thr37/46), 4E-BP (N,O), and GAPDH (loading control) antibodies. (J,K,M,O) Bar-graphs show relative protein expression level calculated after normalization to DMSO control from three or more independent experiments. (P,Q) EGFP-TFEB expressing Neuro2a cells were treated with DMSO/Torin1 (1 µM)/MTP (10 µM)/TFP (10 µM) for 6 h. Images were acquired on high content imaging system and representative images are shown. Scale bar, 10 µm. (Q) Bar-graph showing the percentage EGFP-TFEB nuclear translocation (n = 3). Data information: All data were expressed as means ± SD, one-way ANOVA followed by Dunnett test was used to calculate statistical significance *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant. Source data are available online for this figure.
Figure 5
Figure 5. MTP activates adaptive ER stress and dysregulates ER calcium homeostasis.
(A) Neuro2a cells were treated with MTP (10 µM) for 6 h and mRNA levels of autophagy genes were determined by qRT-PCR. Heatmap shows relative gene expression level after normalization to DMSO-treated control (n = 6). (B,C) Neuro2a cells were treated with DMSO/Tg (1 µM)/MTP (10 µM) for the indicated time points. (B) Protein lysates were immunoblotted for PERK, p-eIF2α, eIF2α, CHOP, and GAPDH (loading control). (C) Bar-graph shows relative expression of p-eIF2α/eIF2α normalized to DMSO control from three independent experiments. (D) mRNA levels of ER stress markers and chaperones were quantified using qRT-PCR. Heatmap depicts relative gene expression normalized to DMSO control, represented as mean (n = 3). (E) Representative Ca2+ imaging trace of experiments where Neuro2a cells were stimulated with 10 µM MTP in absence of extracellular Ca2+ followed by addition of 2 μM thapsigargin (Tg). Here, “n = 113” denotes the number of cells in that particular trace. (F) Representative Ca2+ imaging trace of experiments where cells were stimulated first with 2 μM Tg to deplete ER Ca2+ stores, followed by addition of 10 µM MTP in absence of extracellular Ca2+. Here, “n = 125” denotes the number of cells in that particular trace. (G) Quantitation of MTP (10 μM) induced ER Ca2+ stores depletion before and after the addition of 2 μM Tg. 465 and 355 cells from 5 and 4 independent imaging dishes were analyzed for the two conditions, respectively. (H) Quantitation of Tg (2 μM), induced ER Ca2+ stores depletion before and after the addition of 10 μM MTP. 355 and 465 cells from 4 and 5 independent imaging dishes were analyzed for the two conditions, respectively (“n  =  x, y” where “x” denotes total number of cells imaged and “y” denotes number of traces recorded). Data information: In (G,H), data presented are means ± S.E.M., Unpaired Student t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant. Source data are available online for this figure.
Figure 6
Figure 6. MTP targets JEV replication.
(A) Schematic depicting time course of the JEV infection process (Created with BioRender.com). Viral genome translation initiates between 3–6 h, and with the help of viral nonstructural and host proteins the replication complex (RC) is generated on ER membranes. The dsRNA replicative intermediate is synthesized here, through which several copies of the positive-sense JEV RNA are made. These are used for (i) translation to generate more viral proteins and RCs, (ii) packaging into virus particles that undergo maturation and egress. (B,C) Cortical neurons (B), Neuro2a cells (C), were pre-treated with DMSO/MTP (10 µM) for 1 h, and then infected with JEV (MOI 5) for 1 h in the presence of the drug. Cells were given trypsin treatment to remove dish/cell bound virus particles, and the levels of internalized virus were measured using qRT-PCR. Data is plotted from two independent experiments (n = 6), means ± SD, unpaired Student t-test. (D,F) Neuro2a cells stably expressing EGFP-LC3 were grown on glass coverslips and were mock/JEV (MOI 50) infected. At 1 hpi, cells were given DMSO/MTP treatment for 1 h. Cells were immunostained with JEV envelope antibody, and SIM imaging was peformed. The right panel shows magnified view of the region marked by rectangle. Scale bar, 10 µm, 5 µm (inset). Graph shows quantitation of autophagosome number (green puncta) per cell (E), and JEV particles (red dots) per cell (F), calculated from 15 to 18 cells across two independent coverslips. Quantification was performed using Imaris 8 software and expressed as means ± SEM. Statistical analysis was performed using unpaired Student t-test. (G–I) Neuro2a cells were infected with JEV (MOI 0.1), at 1 hpi, cells were treated with DMSO/MTP (10 µM). Cells were harvested at the indicated hpi. (G) Viral RNA levels were quantified using qRT-PCR. Data represents values obtained from two independent experiments (n = 6), means ± SD, unpaired Student t-test. (H) Cells were treated as described above. Negative strand of viral RNA was quantified by qRT-PCR. Data is plotted from minimum four independent experiments, expressed as means ± SEM, paired Student t-test. (I) Virus titers was determined by plaque assay. Data represents values obtained from two independent experiments (n = 6), means ± SD, unpaired Student t-test. (J–M) Cortical neurons (MOI 1) (J,K), Neuro2a (MOI 1) (L,M), were mock/JEV infected and at 1 hpi, treated with DMSO/MTP (10 µM) (cortical neurons)/TFP (10 µM) (Neuro2a). (J,L) Cells were harvested at the indicated hpi and viral RNA levels were quantified using qRT-PCR. Data represents values obtained from two independent experiments (n = 6). (K,M) Virus titers were measured in culture supernatants using plaque assay, values plotted from two independent experiments (n = 6), means ± SD, unpaired Student t-test. Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant. Source data are available online for this figure.
Figure 7
Figure 7. Antiviral effect of MTP is autophagy-dependent, but does not impact viral RNA translation.
Neuro2a ATG5 KO and ATG7 knockout cell lines were generated by CRISPR-Cas9. (A) Protein lysates of WT and ATG5KO cells analyzed by immunoblotting using ATG5, LC3 (autophagy control), and GAPDH (internal controls) antibodies. (B,C) Neuro2a WT and ATG5 KO cells were infected with JEV (MOI 1). At 1 hpi, cells were treated with MTP at indicated concentrations. Cells were harvested at 24 hpi to measure JEV RNA levels. (B) Relative viral RNA levels after normalization to respective DMSO-treated control. Data is plotted from three independent experiments (n = 6). (C) Culture supernatants were harvested and virus titers was measured using plaque assay, Data is plotted from three independent experiments (n = 9). (D) Protein lysates of WT and ATG7 KO cells analyzed by immunoblotting using ATG7, LC3 (autophagy control), and GAPDH (internal controls) antibodies. (E,F) Neuro2a WT and ATG7 KO cells were infected with JEV (MOI 1). At 1 hpi, cells were treated with MTP at indicated concentrations. Cells were harvested at 24 hpi to measure JEV RNA levels. (E) Relative viral RNA levels after normalization to respective DMSO-treated control. Data is plotted from three independent experiments (n = 9). (F) Culture supernatants were harvested and virus titers were measured using plaque assay, Data is plotted from three independent experiments (n = 9). (G–L) WT and ATG7KO Neuro2a cells were mock/JEV (5 MOI) infected for 1 h, followed by DMSO/MTP (10 µm) treatment till 6 hpi. Cell lysates were prepared from WT (G) and ATG7 KO cells (J), and global polysome profile analysis was performed by the density gradient fractionation system. Polysome-to-monosome (P/M) ratios from two independent experiments are indicated in the respective panel, means ± SD. Percentage distribution of GAPDH mRNA (internal control) (H,K) and viral RNA (I,L) in the monosome and polysome gradient fractions was quantified by qRT-PCR. Similar trends were seen in two independent experiments. Data information: All data are expressed as means ± SD, one-way ANOVA followed by Dunnett test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant (H,I,K,L). Source data are available online for this figure.
Figure 8
Figure 8. MTP induced autophagy reduces inflammatory cytokines secretion and NLRP3 levels in microglial cells.
(A,B) N9 cells were transfected with siNT/ATG5 for 48 h, followed by mock/JEV (MOI 3) infection for 1 h and treatment with MTP (10 µM) till 24 hpi. (A) Cell lysates were prepared and proteins were analyzed by immunoblotting using ATG5, NS3 (infection control), and GAPDH (internal control) antibodies. (B) Culture supernatant was used for the quantitation of cytokine levels using flow cytometry-based CBA assay (n = 3). Two-way ANOVA followed by Tukey test. Similar trends were seen in two independent experiments. (C,D) N9 cells were transfected with siNT/ATG5 for 48 h, then infected with JEV (MOI 3) for 1 h. Post-infection, cells were treated with either DMSO or MTP (10 µM) for 24 h. (C) Cell lysates were analyzed by immunoblotting using NLRP3, ATG5, NS5 (infection control), and GAPDH (internal control) antibodies. (D) Bar-graph shows relative expression of NLRP3/GAPDH normalized to mock control from four independent experiments. Unpaired Students t-test. (E,F) siNT/siATG5 transfected cells were treated with DMSO/LPS (1 µg/ml)/LPS (1 µg/ml) +MTP (10 µM) for 24 h. (E) Cell lysates were analyzed by immunoblotting using NLRP3, ATG5, and GAPDH (internal control) antibodies. (F) Bar-graph shows relative expression of NLRP3/GAPDH normalized to DMSO control from three independent experiments. Unpaired Students t-test. Data information: All data are expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns; non-significant. Source data are available online for this figure.
Figure EV1
Figure EV1. MTP inhibits the secretion of proinflammatory cytokines from JEV infected/LPS-stimulated astrocytes.
(A) Primary astrocytes were isolated from P2 pups, and purity was confirmed through immunofluorescence staining with GFAP antibody. Scale bar, 10 µm. (B) Primary astrocytes were treated with DMSO/MTP (10 µM)/LPS (1 µg/ml)/LPS + MTP for 24 h. Percentage cell viability was measured by MTT assay (n = 3). (C,D) Primary astrocytes were mock/JEV (MOI 1) infected for 1 h, followed by treatment with either DMSO or MTP (10 µM) till 24 h. (C) Viral RNA levels were quantified using qRT-PCR. Graph shows the relative expression levels of JEV RNA normalized to DMSO-treated control. Data is plotted from two independent experiments (n = 6). (D) Culture supernatant was used to determine virus titers using plaque assays. Data represents values obtained from two independent experiments (n = 6). (E,F) Primary astrocytes were infected with JEV (MOI 1) for 1 h then treated with DMSO/MTP (10 µM) till 24 h (E), or were treated with DMSO (mock)/LPS (1 µg/ml)/LPS + MTP for 24 h (F). Culture supernatants were collected and cytokine levels were quantitated by CBA using flow cytometry. Data were analyzed with LEGENDplexTM Multiplex assay software. Data shows values from one representative experiment (n = 3). Similar trends were seen in two independent experiments. Data information: All data expressed as means ± SD, statistical significance was determined using unpaired Student t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure EV2
Figure EV2. Phenothiazines induce functional autophagy flux and do not alter lysosomal pH.
(A,B) EGFP-LC3 expressing stable Neuro2a cells were treated with DMSO/Torin1 (1 µM)/MTP (10 µM)/TFP (10 µM) for 6 h. (A) Representative SIM images are shown. Scale bar, 10 µm. (B) Bar-graph shows quantitation of EGFP-LC3 puncta per cell. Data is acquired from 20 cells across two independent coverslips. (C,D) GFP-LC3-RFP-LC3∆G expressing stable MEFs were treated with DMSO (control)/Torin1 (1 µM)/BafA1 (100 nM) or MTP/TFP (10 µM) for 6 h. (C) Images were visualized by high-content imaging system. Scale bar, 100 µm. (D) Graph showing GFP/RFP ratios (n = 3). (E–H) Neuro2a cells grown on glass coverslips were treated with indicated drugs as described above for 6 h, followed by incubation with 10 µM LysoTracker Red for 40 min (E,F) or 10 µM LysoSensor Yellow-Blue for 5 min (G,H). Representative confocal images are shown. Scale bar, 10 µm (E); 20 µm (G). LysoTracker Red (F) and LysoSensor Yellow-Blue (yellow) (H) fluorescence intensities were calculated from 50 cells across two independent coverslips using ImageJ (Fiji). Data information: All data are normalized to DMSO control and expressed as means ± SD, one-way ANOVA test followed by Dunnett test was used for statistical significance. *P < 0.05; **P < 0.01; ****P < 0.0001.
Figure EV3
Figure EV3. MTP activates adaptive ER stress and dysregulates ER calcium homeostasis.
(A) MEFs were treated with DMSO/MTP (10 µM) for 6 h and mRNA levels of autophagy genes were determined by qRT-PCR. Heatmap shows relative gene expression level after normalization to DMSO-treated control (n = 3). (B,C) MEFs were infected with JEV (MOI 1), at 1 hpi cells were treated with DMSO/Tg (1 µM)/MTP (10 µM) for the indicated time points. (B) Protein lysates were analyzed by immunoblotting using PERK, p-eIF2α, eIF2α, CHOP, NS5 (infection control) and GAPDH (loading control) antibodies. (C) Bar-graph shows relative expression of p-eIF2α/eIF2α normalized to DMSO control from three independent experiments, unpaired Student t-test. (D) MEFs were treated with DMSO/Tg (1 µM)/MTP (10 µM) for the indicated time points. mRNA levels of ER stress markers and chaperones were quantified using qRT-PCR. Heatmap depicts relative gene expression normalized to DMSO control, represented as mean (n = 3). (E) Representative Ca2+ imaging trace of MTP dose–response assay, where “n = 112” denotes the number of cells in that particular trace. Cells were stimulated with increasing doses of MTP- 1 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM followed by addition of 2 μM thapsigargin (Tg) in Ca2+-free buffer. (F) Representative Ca2+ imaging trace of experiments where cells were stimulated with 10 µM MTP in absence of extracellular Ca2+ followed by addition of 2 μM Tg. Here, “n = 85” denotes the number of cells in that particular trace. (G) Representative Ca2+ imaging trace of experiments where cells were stimulated first with 2 μM Tg to deplete ER Ca2+ stores, followed by addition of 10 µM MTP in absence of extracellular Ca2+. Here, “n = 104” denotes the number of cells in that particular trace. (H) Quantitation of MTP (10 μM) induced ER Ca2+ stores depletion before and after the addition of 2 μM Tg. 620 and 539 cells from 9 and 7 independent imaging dishes were analyzed for the two conditions, respectively. (I) Quantitation of Tg, (2 μM) induced ER Ca2+ stores depletion before and after the addition of 10 μM MTP. 539 and 620 cells from 7 and 9 independent imaging dishes were analyzed for the two conditions, respectively (“n  =  x, y” where “x” denotes total number of cells imaged and “y” denotes number of traces recorded). Data presented are mean ± S.E.M., unpaired Student’s t test, **P < 0.01; ***P < 0.001.
Figure EV4
Figure EV4. MTP induced ER stress is essential for autophagy and antiviral effect.
(A) Neuro2a cells were treated with DMSO/Tg (1 µM)/MTP (10 µM)/4-PBA (2 mM)/4-PBA (2 mM) + MTP (10 µM) for 24 h. MTT assay was used to calculate % cell viability, and normalized to DMSO treated control (n = 3). (B,C) Neuro2a cells were treated with DMSO/Tg (1 µM)/MTP (10 µM)/4-PBA (2 mM)/4-PBA (2 mM) + MTP (10 µM) for 6 h. (B) Protein lysates were analyzed by immunoblotting using LC3 and GAPDH (loading control) antibodies. (C) Bar-graph shows relative protein expression level of LC3II/GAPDH calculated after normalization to DMSO control. Values were plotted from three independent experiments. (D,E) Neuro2a cells were infected with JEV at MOI 1 for 1 h. Post-infection, cells were treated with DMSO/Tg (1 µM)/MTP (10 µM)/4-PBA (2 mM) + MTP (10 µM) for 24 h. (D) Cells were harvested, viral transcript levels were measured using qRT-PCR and normalized to DMSO-treated infected control from two independent experiments (n = 6). (E) Virus titers was measured in culture supernatant using plaque assay, value plotted from two independent experiments (n = 6). Data information: All data were expressed as means ± SD, unpaired Student t-test was used to calculate statistical significance *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
Figure EV5
Figure EV5. Antiviral effect of phenothiazines is autophagy dependent.
(A,B) MEFs were mock/JEV infected (MOI 1) and at 1 hpi, treated with DMSO/MTP (10 µM). (A) Cells were harvested at the indicated hpi and viral RNA levels were quantified using qRT-PCR. Data represents values obtained from two independent experiments (n = 6). (B) Culture supernatant was used to determine virus titers using plaque assay. Data is plotted from two independent experiments (n = 6), and compared by unpaired Student t-test. (C) WT and ATG5 KO MEFs were treated with indicated concentrations of MTP for 24 h, and the percentage cell viability was measured and normalized to respective DMSO-treated controls (n = 3). (D,E) WT and ATG5 KO MEFs were infected with JEV at MOI 1, and at 1 hpi treated with MTP at indicated concentrations. Cells were harvested at 24 hpi and the relative viral RNA levels were quantitated using qRT-PCR, and plotted after normalization to respective DMSO-treated control. Data represents values from three independent experiments (n = 9). (E) Culture supernatant was collected and virus titers was determined using plaque assay. Data is plotted from three independent experiments (n = 9). (F–K) WT and ATG5 KO MEFs were mock/JEV (5 MOI) infected for 1 h, followed by DMSO/MTP (10 µm) treatment till 6 hpi. (F,I) Global polysome profile analysis of cell lysates were performed by the density gradient fractionation system. Polysome-to-monosome (P/M) ratios from two independent experiments, means ± SD. (G,H,J,K) Percentage distribution of GAPDH mRNA (housekeeping gene) (G,J), viral RNA (H,K) in the monosome and polysome fractions was analyzed by qRT-PCR. Similar trends were seen in two independent experiments. Data information: One-way ANOVA followed by Dunnett test was used for the determination of statistical significance, *P < 0.05; **P < 0.01; ****P < 0.0001, ns, non-significant.
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