Journal: bioRxiv

Article Title: A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis

doi: 10.1101/2024.09.09.611003

Figure Lengend Snippet: (A-D) Immunofluorescence imaging displayed BrdU (green) and nuclear staining with propidium iodide (PI) (red) in MRC5 human lung fibroblasts. A dose- dependent increase in BrdU incorporation was observed in MRC5 cells stimulated with increasing concentrations of noradrenaline (NA), peaking at 25 µM (D, P = 0.0159). This response was reversed upon co-incubation with terazosin (E, P = 0.0441). (F) MTT assays demonstrated a significant reduction in the number of viable cells in MRC5 human lung fibroblasts and IPF fibroblasts when stimulated with NA and treated with terazosin ( P = 0.0493 and P = 0.0156, respectively). This effect was not observed in normal human lung fibroblasts, indicating that IPF fibroblasts are poised to receive and respond to noradrenergic signals via an α1-AR dependent mechanism. (G-I) Immunofluorescence imaging demonstrated expression of ADRA1D (red) and α-SMA (green), with nuclear staining by DAPI (blue) in human precision-cut lung slices. Following exposure to a fibrotic cocktail, a marked increase in α-SMA (ACTA2) expression was observed in stromal cells adjacent to alveoli, with some cells showing co- expression of ADRA1D (white arrows, G, H). This expression pattern was consistent with trichrome staining and matched ACTA2 expression quantified by PCR analysis (J, P = 0.0236). The introduction of terazosin to the culture media reversed these fibrotic effects (I, J, P = 0.0106). Images were captured at 20x magnification. Data are presented as mean ± SEM or median ± IQR, with statistical analysis performed using Student’s t-test for normally distributed data and Mann-Whitney or Kruskal-Wallis tests with Dunn’s multiple comparisons for non- normally distributed data. * P < 0.05, ** P < 0.01. ADRA1D, α1-adrenoreceptor subtype D; α-SMA, alpha-smooth muscle actin; BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; FC, fibrotic cocktail; IPF, idiopathic pulmonary fibrosis; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NA, noradrenaline; NHLF, normal human lung fibroblast; PCR, polymerase chain reaction; PI, propidium iodide; TZ, terazosin.

Article Snippet: MRC5 fibroblasts, normal human lung fibroblasts, and IPF fibroblasts were procured from ATCC (Manassas, VA), Lonza (Allendale, NJ), and Asterand Bioscience (Detroit, MI), respectively.

Techniques: Immunofluorescence, Imaging, Staining, BrdU Incorporation Assay, Incubation, Expressing, MANN-WHITNEY, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis

doi: 10.1101/2024.09.09.611003

Figure Lengend Snippet: (A-D) Immunofluorescence imaging displayed BrdU (green) and nuclear staining with propidium iodide (PI) (red) in MRC5 human lung fibroblasts. A dose- dependent increase in BrdU incorporation was observed in MRC5 cells stimulated with increasing concentrations of noradrenaline (NA), peaking at 25 µM (D, P = 0.0159). This response was reversed upon co-incubation with terazosin (E, P = 0.0441). (F) MTT assays demonstrated a significant reduction in the number of viable cells in MRC5 human lung fibroblasts and IPF fibroblasts when stimulated with NA and treated with terazosin ( P = 0.0493 and P = 0.0156, respectively). This effect was not observed in normal human lung fibroblasts, indicating that IPF fibroblasts are poised to receive and respond to noradrenergic signals via an α1-AR dependent mechanism. (G-I) Immunofluorescence imaging demonstrated expression of ADRA1D (red) and α-SMA (green), with nuclear staining by DAPI (blue) in human precision-cut lung slices. Following exposure to a fibrotic cocktail, a marked increase in α-SMA (ACTA2) expression was observed in stromal cells adjacent to alveoli, with some cells showing co- expression of ADRA1D (white arrows, G, H). This expression pattern was consistent with trichrome staining and matched ACTA2 expression quantified by PCR analysis (J, P = 0.0236). The introduction of terazosin to the culture media reversed these fibrotic effects (I, J, P = 0.0106). Images were captured at 20x magnification. Data are presented as mean ± SEM or median ± IQR, with statistical analysis performed using Student’s t-test for normally distributed data and Mann-Whitney or Kruskal-Wallis tests with Dunn’s multiple comparisons for non- normally distributed data. * P < 0.05, ** P < 0.01. ADRA1D, α1-adrenoreceptor subtype D; α-SMA, alpha-smooth muscle actin; BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; FC, fibrotic cocktail; IPF, idiopathic pulmonary fibrosis; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NA, noradrenaline; NHLF, normal human lung fibroblast; PCR, polymerase chain reaction; PI, propidium iodide; TZ, terazosin.

Article Snippet: MRC5 fibroblasts, normal human lung fibroblasts, and IPF fibroblasts were procured from ATCC (Manassas, VA), Lonza (Allendale, NJ), and Asterand Bioscience (Detroit, MI), respectively.

Techniques: Immunofluorescence, Imaging, Staining, BrdU Incorporation Assay, Incubation, Expressing, MANN-WHITNEY, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis

doi: 10.1101/2024.09.09.611003

Figure Lengend Snippet: (A-D) Immunofluorescence imaging displayed BrdU (green) and nuclear staining with propidium iodide (PI) (red) in MRC5 human lung fibroblasts. A dose- dependent increase in BrdU incorporation was observed in MRC5 cells stimulated with increasing concentrations of noradrenaline (NA), peaking at 25 µM (D, P = 0.0159). This response was reversed upon co-incubation with terazosin (E, P = 0.0441). (F) MTT assays demonstrated a significant reduction in the number of viable cells in MRC5 human lung fibroblasts and IPF fibroblasts when stimulated with NA and treated with terazosin ( P = 0.0493 and P = 0.0156, respectively). This effect was not observed in normal human lung fibroblasts, indicating that IPF fibroblasts are poised to receive and respond to noradrenergic signals via an α1-AR dependent mechanism. (G-I) Immunofluorescence imaging demonstrated expression of ADRA1D (red) and α-SMA (green), with nuclear staining by DAPI (blue) in human precision-cut lung slices. Following exposure to a fibrotic cocktail, a marked increase in α-SMA (ACTA2) expression was observed in stromal cells adjacent to alveoli, with some cells showing co- expression of ADRA1D (white arrows, G, H). This expression pattern was consistent with trichrome staining and matched ACTA2 expression quantified by PCR analysis (J, P = 0.0236). The introduction of terazosin to the culture media reversed these fibrotic effects (I, J, P = 0.0106). Images were captured at 20x magnification. Data are presented as mean ± SEM or median ± IQR, with statistical analysis performed using Student’s t-test for normally distributed data and Mann-Whitney or Kruskal-Wallis tests with Dunn’s multiple comparisons for non- normally distributed data. * P < 0.05, ** P < 0.01. ADRA1D, α1-adrenoreceptor subtype D; α-SMA, alpha-smooth muscle actin; BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; FC, fibrotic cocktail; IPF, idiopathic pulmonary fibrosis; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NA, noradrenaline; NHLF, normal human lung fibroblast; PCR, polymerase chain reaction; PI, propidium iodide; TZ, terazosin.

Article Snippet: MRC5 fibroblasts, normal human lung fibroblasts, and IPF fibroblasts were procured from ATCC (Manassas, VA), Lonza (Allendale, NJ), and Asterand Bioscience (Detroit, MI), respectively.

Techniques: Immunofluorescence, Imaging, Staining, BrdU Incorporation Assay, Incubation, Expressing, MANN-WHITNEY, Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Human coronaviruses activate and hijack the host transcription factor HSF1 to enhance viral replication

doi: 10.1007/s00018-024-05370-5

Figure Lengend Snippet: Human coronaviruses induce HSF1 phosphorylation and DNA-binding activity. A The human coronavirus lipid bilayer comprising the spike protein (blue), the membrane protein (orange) and the envelope protein (green), and the viral RNA (purple) associated with the nucleocapsid protein (pink) are shown. B Schematic representation of genome structure, classification and receptors of the human coronaviruses HCoV-229E, HCoV-NL63 and HCoV-OC43. ORF1a and ORF1b are represented as light blue boxes; genes encoding structural proteins spike (S), nucleocapsid (N), envelope (E), membrane (M), and hemagglutinin-esterase (HE) and genes encoding accessory proteins are shown. hAPN, human aminopeptidase N; 9-O-Ac-Sia, N-acetyl-9-O-acetylneuraminic acid; ACE2, angiotensin-converting enzyme 2. C Immunoblot (IB) analysis of pHSF1-Ser326, HSF1, viral nucleocapsid (N) and β-actin protein levels in MRC-5 and Caco-2 hACE2 cells mock-infected (−) or infected (+) with HCoV-229E or HCoV-OC43 (MRC-5) for 24 h, or HCoV-NL63 (Caco-2 hACE2) for 72 h at a m.o.i. of 0.1 TCID 50 /cell. D , E Whole-cell extracts (WCE) from samples mock-infected (Mock) or infected with HCoV-229E (1 TCID 50 /cell) were analyzed for pHSF1-Ser326, HSF1, N and α-tubulin protein levels at early ( D ) or late ( E ) times post infection (p.i.) by IB. F Schematic representation of HSF1 domain organization: DBD, DNA Binding Domain; HR-A/HR-B, Heptad Repeats A and B; RD, Regulatory Domain; HR-C, Heptad Repeat C; AD, Activation Domain. Phosphorylation sites Ser121, Ser303, and Ser326 are shown. G MRC-5 cells were treated with bortezomib (BTZ, 20 nM) for 16 h, exposed to heat stress (HS, 43 °C, 40 min), mock-infected (Mock) or infected with HCoV-229E (0.1 TCID 50 /cell) for 40 h. WCE were analyzed for levels of HSF1-Ser326, -Ser303 and -Ser121 phosphorylation, HSF1, viral N and α-tubulin proteins by IB (top panels). In the same samples, HSF1 DNA-binding activity was analyzed by EMSA (bottom panel). Positions of the HSF DNA-binding complex (HSF), constitutive HSE-binding activity (CHBA) and nonspecific protein-DNA interaction (NS) are shown. H MRC-5 cells were mock-infected or infected with HCoV-229E (1 TCID 50 /cell). At different times p.i., HSF1 DNA-binding activity was analyzed by EMSA. I WCE from samples infected with HCoV-229E (0.1 TCID 50 /cell, 40 h p.i.) were preincubated with different dilutions of anti-HSF1 or anti-HSF2 antibodies and analyzed by gel mobility supershift assay. The position of the nonsupershifted virus-induced HSF1 complex is indicated at the left (No Ab)

Article Snippet: Human normal lung MRC-5 fibroblasts (American Type Culture Collection, ATCC, CCL-171), African green monkey kidney Vero cells (ATCC, CCL-81), Vero E6 cells (ATCC, CRL-1586), HeLa cells and Caco-2 cells (ATCC) were grown at 37 °C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Vero, Vero E6, Caco-2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Binding Assay, Activity Assay, Membrane, Western Blot, Infection, Activation Assay, Virus

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Human coronaviruses activate and hijack the host transcription factor HSF1 to enhance viral replication

doi: 10.1007/s00018-024-05370-5

Figure Lengend Snippet: HCoV infection triggers HSF1 nuclear translocation in human lung cells. A Schematic representation of HSF1 intracellular localization under physiological (no stress) and stress conditions. B Immunoblot analysis of pHSF1-Ser326, HSF1 and viral spike (S) protein levels in cytoplasmic (Cyt) and nuclear (Nu) fractions of MRC-5 cells mock-infected (−) or infected (+) with HCoV-229E (0.1 TCID 50 /cell) for 24 h. Antibodies against α-tubulin and histone H3 (Hist-H3) were used as loading controls for cytoplasmic and nuclear fractions, respectively. C Confocal images of pHSF1-Ser326 (red) and α-tubulin (green) intracellular localization in MRC-5 cells mock-infected or infected with HCoV-229E (1 TCID 50 /cell) at 30 h p.i.. Nuclei are stained with Hoechst (blue). Merge and zoom images are shown. Scale bar, 20 μm (zoom, 5 μm). D Confocal 3D-reconstruction of pHSF1-Ser326 (red) intranuclear localization in MRC-5 cells mock-infected or infected as in C; α-tubulin is shown in green. Nuclei are stained with Hoechst (blue). The overlay of the fluorochromes is shown. E Confocal images of pHSF1-Ser326 (red) and α-tubulin (green) intracellular localization in MRC-5 cells mock-infected or infected with HCoV-OC43 (1 TCID 50 /cell) at 30 h p.i.. Nuclei are stained with Hoechst (blue). Merge images are shown. Scale bar, 20 μm. F IB of pHSF1-Ser326, HSF1, N and β-actin protein levels in MRC-5 cells mock-infected or infected with HCoV-OC43 (0.1 TCID 50 /cell) for 24 h (left panels). HSF1 monomers and trimers in the same samples are shown (right panel)

Article Snippet: Human normal lung MRC-5 fibroblasts (American Type Culture Collection, ATCC, CCL-171), African green monkey kidney Vero cells (ATCC, CCL-81), Vero E6 cells (ATCC, CRL-1586), HeLa cells and Caco-2 cells (ATCC) were grown at 37 °C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Vero, Vero E6, Caco-2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Infection, Translocation Assay, Western Blot, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Human coronaviruses activate and hijack the host transcription factor HSF1 to enhance viral replication

doi: 10.1007/s00018-024-05370-5

Figure Lengend Snippet: HCoV infection turns on an HSF1-driven transcriptional program in human lung cells. A – D Expression profile of selected HSF1-target genes affected by HCoV-229E infection (0.1 TCID 50 /cell) for 24 h in MRC-5 cells relative to mock-infected cells as determined by qRT-PCR array (PAHS-076ZD-2-Qiagen). Heat Map ( A ) and Volcano plot ( B ) of 84 human HSPs and chaperones/cochaperones gene expression. In A each row represents a single gene, each column represents a sample [mock-infected or HCoV-229E infected cells (229E); n = 3]. The gradual color ranging from blue to red represents the mRNA expression level (Z-score). In the Volcano plot ( B ) fold regulation threshold is set to 2 and p -value cut off is 0.05; each dot represents a gene: red dots indicate significantly upregulated genes and blue dots indicate significantly downregulated genes. Selected HSPs and chaperones/cochaperones genes whose expression is highly induced by HCoV infection are shown in C ; levels of heat shock factors (HSF1, HSF2 and HSF4) gene expression affected by HCoV infection are shown in D . E Expression of non-canonical HSF1-target genes AIRAP, COX-2 and NKRF in samples treated as in A as determined by qRT-PCR. Error bars indicate means ± S.D. (n = 3). *p < 0.05; Student’s t -test ( D , E ). F Levels of HSP90, GRP94, GRP78, HSP70, HSPA6, HSP60, AIRAP, viral spike (S) and β-actin proteins were determined by IB in MRC-5 cells mock-infected or infected with HCoV-229E (1 TCID 50 /cell) at different times p.i.. G Schematic representation of the puromycin-labeling experimental protocol. H MRC-5 cells were mock-infected (−) or infected with HCoV-229E (+) (1 TCID 50 /cell), or treated with vehicle (−) or cycloheximide (CHX, 100 µg/ml, +) for 3 h, as positive control of translation inhibition. At different times p.i., puromycin (2.5 µg/ml, +) was added thirty minutes before harvesting and IB analysis. Blot membranes were stained with Ponceau S solution to assess the steady-state proteomes (top panel) and then hybridized with anti-puromycin antibodies to detect de novo synthesized nascent polypeptides (middle panel). HCoV-N protein is indicated by red arrowheads. Levels of GRP94, GRP78, HSP70, HCoV-N and β-actin proteins detected by IB in the same samples are shown (bottom panels)

Article Snippet: Human normal lung MRC-5 fibroblasts (American Type Culture Collection, ATCC, CCL-171), African green monkey kidney Vero cells (ATCC, CCL-81), Vero E6 cells (ATCC, CRL-1586), HeLa cells and Caco-2 cells (ATCC) were grown at 37 °C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Vero, Vero E6, Caco-2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Infection, Expressing, Quantitative RT-PCR, Labeling, Positive Control, Inhibition, Staining, Synthesized

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Human coronaviruses activate and hijack the host transcription factor HSF1 to enhance viral replication

doi: 10.1007/s00018-024-05370-5

Figure Lengend Snippet: Effect of different SARS-CoV-2 variants on HSF1 activation and HSP70 expression in the host cell. A Immunoblot analysis of SARS-CoV-2 viral spike, pHSF1-Ser326, HSF1 and α-tubulin protein levels in Vero-hACE2-TMPRSS2 cells mock-infected or infected for 48 h with SARS-CoV-2 Wuhan strain (0.1 PFU/cell), or in Vero E6 cells exposed to heat-stress (HS, 43 °C, 40 min). B Schematic representation of the experimental design of SARS-CoV-2 variants infection of Vero-hACE2-TMPRSS2 or MRC5-hACE2 cells. Immunoblot analysis of human angiotensin-converting enzyme 2 (hACE2) protein levels in MRC-5 wild type or expressing the human ACE2-receptor (M-hACE2) is shown. C Vero-hACE2-TMPRSS2 cells were mock-infected or infected with Wuhan, Alpha, Delta (0.1 PFU/cell) or Omicron (0.5 PFU/cell) SARS-CoV-2 variants for 48 h. Equal amounts of whole-cell extracts (10 μl) were analyzed for levels of spike, HSF1-Ser326, HSF1 and β-actin by IB (top panels). The pHSF1/HSF1 ratio was determined after normalizing to β-actin and expressed as fold induction of the mock-infected control, which was arbitrarily set to 1 (bottom panel). D MRC5-hACE2 cells were mock-infected or infected with the SARS-CoV-2 Omicron BA.1 variant (0.1 PFU/cell) for 24 h. Equal amounts of whole-cell extracts (15 μl) were analyzed for levels of spike, HSF1-Ser326, HSF1 and β-actin (left panels). The pHSF1/HSF1 ratio was determined after normalizing to β-actin and expressed as fold induction of the mock-infected control, which was arbitrarily set to 1 (right panel). E , F Vero-hACE2-TMPRSS2 cells were mock-infected or infected with different SARS-CoV-2 variants as in C . Equal amounts of whole-cell extracts (20 μl) were analyzed for levels of spike, GRP94, HSP90, HSP70, HSP60, pHSF1-Ser326, HSF1 and α-tubulin by IB ( E ). Uncleaved S proteins (S0) and S1 subunits are indicated by arrows. Relative amounts of GRP94, HSP90, HSP70, HSP60 and pHSF1 were determined after normalizing to α-tubulin and expressed as fold induction of the mock-infected control, which was arbitrarily set to 1 ( F ). C , D , F n = 4 (two technical replicates over two biological replicates)

Article Snippet: Human normal lung MRC-5 fibroblasts (American Type Culture Collection, ATCC, CCL-171), African green monkey kidney Vero cells (ATCC, CCL-81), Vero E6 cells (ATCC, CRL-1586), HeLa cells and Caco-2 cells (ATCC) were grown at 37 °C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Vero, Vero E6, Caco-2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Activation Assay, Expressing, Western Blot, Infection, Control, Variant Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Human coronaviruses activate and hijack the host transcription factor HSF1 to enhance viral replication

doi: 10.1007/s00018-024-05370-5

Figure Lengend Snippet: HSF1 is required for efficient HCoV replication. A Immunoblot of pHSF1-Ser326, HSF1, AIRAP, viral spike (S), α-tubulin and GAPDH protein levels in MRC-5 cells transiently transfected with two different HSF1-siRNAs [siHSF1 1 (left) and siHSF1 2 (right); +] or scramble-RNA (−) for 48 h, and infected with HCoV-229E (0.1 TCID 50 /cell) or mock infected (Mock) for 24 h. B The relative amount of total HSF1, AIRAP and viral S protein, normalized to the loading control in the same sample, were determined by densitometric analysis using ImageJ software. Error bars indicate means ± S.D. (n = 3). C In parallel, virus yield from supernatants of infected cells was determined at 24 h p.i. by TCID 50 infectivity assay. Data, expressed as percentage of control, represent the mean ± S.D. (n = 3). *p < 0.05, **p < 0.01; Student’s t -test ( B , C ). D Schematic representation of HCoV genomic RNA transfection assays. E–H Wild-type (wt) or stably HSF1-silenced (HSF1i) HeLa cells were co-transfected with HCoV-229E (229E gRNA) or HCoV-OC43 (OC43 gRNA) genomic RNA and the pCMV-GFP vector for 4 h. After 48 h (229E) or 72 h (OC43), levels of HSF1, viral spike (S) and nucleocapsid (N), GFP and GAPDH proteins were analyzed by IB ( E , G ). In parallel, virus yield in the supernatant of transfected cells was determined by TCID 50 infectivity assay ( F , H ). Data, expressed as TCID 50 /ml, represent the mean ± S.D. (n = 3). *p < 0.05; Student’s t -test

Article Snippet: Human normal lung MRC-5 fibroblasts (American Type Culture Collection, ATCC, CCL-171), African green monkey kidney Vero cells (ATCC, CCL-81), Vero E6 cells (ATCC, CRL-1586), HeLa cells and Caco-2 cells (ATCC) were grown at 37 °C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Vero, Vero E6, Caco-2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Western Blot, Transfection, Infection, Control, Software, Virus, Stable Transfection, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Human coronaviruses activate and hijack the host transcription factor HSF1 to enhance viral replication

doi: 10.1007/s00018-024-05370-5

Figure Lengend Snippet: Inhibition of virus-induced HSF1 activation by DTHIB impairs HCoV replication. A Structure of DTHIB (Direct Targeted HSF1 Inhibitor). B MRC-5 cells mock-infected or infected with HCoV-229E (0.1 TCID 50 /cell) were treated with different concentrations of DTHIB immediately after the adsorption period. Virus yield (Ο) was determined at 24 h p.i. by TCID 50 infectivity assay. Data, expressed as TCID 50 /ml, represent the mean ± S.D. (n = 4). *p < 0.05, **p < 0.01; ANOVA test. In parallel, cell viability (△) was determined in mock-infected cells by MTT assay. Absorbance (O.D.) of converted dye was measured at λ = 570 nm. C Immunoblot of pHSF1-Ser326, HSF1, viral N and α-tubulin protein levels in samples treated as in B. D Immunoblot analysis of HSF1 levels in cytoplasmic (Cytosol) and nuclear (Nucleus) fractions of MRC-5 cells mock-infected (−) or infected ( +) with HCoV-229E (0.1 TCID 50 /cell) for 24 h and treated with DTHIB (10 µM, +) or vehicle (−) immediately after the adsorption period (left panels). Antibodies against α-tubulin and Histone H3 (H3) were used as a loading control for cytoplasmic and nuclear fractions, respectively. Virus yield was determined at 24 h p.i. by TCID 50 infectivity assay (right panel). Data, expressed as TCID 50 /ml, represent the mean ± S.D. (n = 4). *p < 0.05; Student’s t -test. E Confocal images of pHSF1-Ser326 (red) and α-tubulin (green) intracellular localization in MRC-5 cells mock-infected or infected with HCoV-229E (1 TCID 50 /cell) for 30 h and treated with DTHIB (5 µM) or control diluent after the adsorption period. Nuclei are stained with Hoechst (blue). Merge images are shown. Scale bar, 20 µm. F MRC-5 cells mock-infected or infected with HCoV-229E (0.1 TCID 50 /cell) were treated with 10 μM DTHIB (filled bars) or control vehicle ( C , empty bar) at 3 h before infection (PRE), immediately after the adsorption period (0 h), at 10 h after infection (10 h p.i.), or only during the adsorption period (ADS). Virus yield was determined at 24 h p.i. by TCID 50 assay. Data, expressed as TCID 50 /ml, represent the mean ± S.D. (n = 3). *p < 0.05; ANOVA test. G Immunoblot of pHSF1-Ser326, HSF1, AIRAP, HCoV-N and α-tubulin protein levels of samples treated as in F . H , I MRC-5 cells were mock-infected or infected with HCoV-229E (1 TCID 50 /cell) and treated with DTHIB (7.5 µM). At different times p.i., virus yield was determined by TCID 50 infectivity assay. Data, expressed as TCID 50 /ml, represent the mean ± S.D. (n = 3). *p < 0.05, **p < 0.01; Student’s t -test (H). In parallel, levels of pHSF1-Ser326, HSF1, HSP70, HSPA6, HSP60, AIRAP, HCoV-N and α-tubulin proteins were determined by IB ( I ). J Confocal images of viral nucleoprotein (red) and dsRNA (green) intracellular localization in MRC-5 cells infected with HCoV-229E (1 TCID 50 /cell) for 30 h and treated with DTHIB (5 µM) or control diluent immediately after the adsorption period. Nuclei are stained with Hoechst (blue). Merge and zoom images are shown. Scale bar, 20 µm (zoom, 7 µm). K Levels of M-229E mRNA were analyzed by qRT-PCR in samples treated as in J. Error bars indicate means ± S.D. (n = 3). *p < 0.05; Student’s t -test. L MRC5-hACE2 cells infected with SARS-CoV-2 Omicron BA.1 variant (0.1 PFU/cell) were treated with different concentrations of DTHIB immediately after the adsorption period. Virus yield was determined at 22 h p.i. by plaque assay. Data, expressed as PFU/ml, represent the mean ± S.D. of samples from three independent experiments. ***p < 0.001; **p < 0.01; ANOVA test

Article Snippet: Human normal lung MRC-5 fibroblasts (American Type Culture Collection, ATCC, CCL-171), African green monkey kidney Vero cells (ATCC, CCL-81), Vero E6 cells (ATCC, CRL-1586), HeLa cells and Caco-2 cells (ATCC) were grown at 37 °C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Vero, Vero E6, Caco-2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Inhibition, Virus, Activation Assay, Infection, Adsorption, MTT Assay, Western Blot, Control, Staining, Quantitative RT-PCR, Variant Assay, Plaque Assay

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: A. RT-qPCR of ITPR1 , ITPR2 and ITPR3 genes at day 3 after treatment of MRC5-MYC:ER cells with 4OHT. Mean +/- SEM of n = 5 independent experiments. Two-Way ANOVA. P-values are indicated. B. RT-qPCR of ITPR1 , BIM and PUMA genes at the indicated times after treatment of MRC5-MYC:ER cells with 4OHT. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated. C. ChIP-seq analysis of MYC occupancy at the ITPR1 promoter region in MYC-overexpressing cells (U2OS, HeLa) (GSE44672).

Article Snippet: MRC5 normal human embryonic lung fibroblasts, U2OS cancer cells (ATCC, Manassas, VA, USA) and virus-producing cells 293-GP and 293-T (Clontech) were cultured in Dulbecco′s modified Eagle′s medium (DMEM, Life Technologies) with GlutaMax supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin/streptomycin (Life Technologies).

Techniques: Quantitative RT-PCR, ChIP-sequencing

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: MRC5-MYC:ER cells were transfected with a control siRNA pool (siControl) or with a siRNA pool targeting ITPR1 (siITPR1) and then treated or not with 4OHT. A. RT-qPCR of ITPR1 gene at day 3 after 4OHT treatment. Mean +/- SEM of n = 5 independent experiments. Two-Way ANOVA. P-values are indicated. B. Western blot showing ITPR1 protein levels at day 3 after 4OHT treatment and α-Tubulin protein levels as loading control. Representative images of n = 3 independent experiments. C . Crystal violet staining at day 10 after 4OHT treatment. Representative image of n = 3 independent experiments. D . Percentage of trypan blue-positive cells counted at day 3 after 4OHT treatment. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated. E . Cell death monitored using SYTOX Green in the first 40h after 4OHT treatment. Representative experiment from n=3 independent experiments.

Article Snippet: MRC5 normal human embryonic lung fibroblasts, U2OS cancer cells (ATCC, Manassas, VA, USA) and virus-producing cells 293-GP and 293-T (Clontech) were cultured in Dulbecco′s modified Eagle′s medium (DMEM, Life Technologies) with GlutaMax supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin/streptomycin (Life Technologies).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Staining

Journal: bioRxiv

Article Title: MYC shapes ER-mitochondria calcium transfer by directly targeting ITPR1 : implications for MYC-induced safeguard mechanisms and cancer

doi: 10.1101/2024.08.28.610025

Figure Lengend Snippet: A. MRC5-MYC:ER cells were transfected with a control siRNA pool (siControl) or with a siRNA pool targeting ITPR1 (siITPR1) or VDAC3 (siVDAC3) and then treated or not with 4OHT. RT-qPCR of ITPR1 gene (left) or VDAC3 gene (right) at day 3 after 4OHT treatment. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated. B . MRC5-MYC:ER cells overexpressing mito-GEM-GECO1 ratiometric mitochondrial Ca 2+ gene reporter were transfected with a control siRNA pool (siControl) or with a siRNA pool targeting ITPR1 (siITPR1) or VDAC3 (siVDAC3) and then treated or not with 4OHT for 2 days. Resting mitochondrial Ca 2+ levels according to the ratio F(437-499)/F(520-755) were measured. Representative images are shown as well as quantitative results from n = 3 independent experiments. siControl -4OHT: n = 148 cells, siITPR1 -4OHT: n = 129 cells, siVDAC3 - 4OHT: n = 198 cells, siControl +4OHT: n = 122 cells, siITPR1 +4OHT: n = 152 cells, siVDAC3 +4OHT: n = 159 cells. Mean ± SEM are shown. Two-way ANOVA test. P-values are indicated. C . Percentage of trypan blue-positive cells counted in MRC5-MYC:ER cells transfected with siControl, siITPR1 or siVDAC3 and then treated or not with 4OHT for 3 days. Mean +/- SEM of n = 3 independent experiments. Two-Way ANOVA. P-values are indicated.

Article Snippet: MRC5 normal human embryonic lung fibroblasts, U2OS cancer cells (ATCC, Manassas, VA, USA) and virus-producing cells 293-GP and 293-T (Clontech) were cultured in Dulbecco′s modified Eagle′s medium (DMEM, Life Technologies) with GlutaMax supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin/streptomycin (Life Technologies).

Techniques: Transfection, Control, Quantitative RT-PCR