human normal lung mrc 5 fibroblasts  (ATCC)

Journal: bioRxiv

Article Title: Human coronaviruses activate and hijack the proteostasis guardian HSF1 to enhance viral replication

doi: 10.1101/2022.12.22.519205

Figure Lengend Snippet: ( 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 24h, or HCoV-NL63 (Caco-2 hACE2) for 72h 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 16h, exposed to heat stress (HS, 43°C, 40 min), mock-infected (Mock) or infected with HCoV-229E (0.1 TCID 50 /cell) for 40h. 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, 40h 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), Caco-2 hACE2 cells, stably expressing the hACE2 receptor, and HeLa cells, stably transfected with pSUPER-HSF1i/pcDNA (HeLa-HSF1i) or control (HeLa wild-type) plasmids, were grown at 37°C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Caco-2 hACE2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

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

Journal: bioRxiv

Article Title: Human coronaviruses activate and hijack the proteostasis guardian HSF1 to enhance viral replication

doi: 10.1101/2022.12.22.519205

Figure Lengend Snippet: ( A ) Schematic representation of HSF1 intracellular localization under physiological (no stress) and stress condition. ( 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 24h. Antibodies against α-tubulin (α-tub) 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 30h 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 30h 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 TCID50/cell) for 24h (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), Caco-2 hACE2 cells, stably expressing the hACE2 receptor, and HeLa cells, stably transfected with pSUPER-HSF1i/pcDNA (HeLa-HSF1i) or control (HeLa wild-type) plasmids, were grown at 37°C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Caco-2 hACE2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Western Blot, Infection, Staining

Journal: bioRxiv

Article Title: Human coronaviruses activate and hijack the proteostasis guardian HSF1 to enhance viral replication

doi: 10.1101/2022.12.22.519205

Figure Lengend Snippet: ( A-D ) Expression profile of selected HSF1-target genes affected by HCoV-229E infection (0.1 TCID50/cell) for 24h in MRC-5 cells relative to mock-infected cells as determined by microarray analysis. Heat Map (A) and Volcano plot (B) of 84 human heat shock protein and chaperones/co-chaperones gene expression. In (A) each row represents a single gene, each column represents a sample [mock-infected (Mock) or HCoV-229E infected cells; n=3]. The gradual color ranging from blue to red represents the mRNA expression level. 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 heat shock proteins and chaperones/co-chaperones 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. * = 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 immunoblot in MRC-5 cells mock-infected or infected with HCoV-229E (1 TCID 50 /cell) at different times p.i.

Article Snippet: Human normal lung MRC-5 fibroblasts (American Type Culture Collection, ATCC, CCL-171), Caco-2 hACE2 cells, stably expressing the hACE2 receptor, and HeLa cells, stably transfected with pSUPER-HSF1i/pcDNA (HeLa-HSF1i) or control (HeLa wild-type) plasmids, were grown at 37°C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Caco-2 hACE2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Expressing, Infection, Microarray, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Human coronaviruses activate and hijack the proteostasis guardian HSF1 to enhance viral replication

doi: 10.1101/2022.12.22.519205

Figure Lengend Snippet: (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 48h, and infected with HCoV-229E (0.1 TCID 50 /cell) or mock-infected (Mock) for 24h (top panels). Virus yield was determined at 24h p.i. by TCID 50 infectivity assay (bottom panel). Data, expressed as percentage of control, represent the mean ± S.D. of duplicate samples. * = p < 0.05; Student’s t -test. ( B ) Wild type (wt) or stably HSF1-silenced (HSF1i) HeLa cells were co-transfected with HCoV-229E genomic RNA (229E gRNA) and the pCMV-GFP vector for 4h. After 48h, levels of HSF1, viral spike (S) and nucleocapsid (N), GFP and GAPDH proteins were analyzed by IB (top panels). Virus yield in the supernatant of transfected cells was determined by TCID50 infectivity assay (bottom panel). Data, expressed as TCID50/ml, represent the mean ± S.D. of duplicate samples. * = p < 0.05; Student’s t -test. ( C ) Structure of DTHIB (Direct Targeted HSF1 Inhibitor). ( D ) 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 ( O ) was determined at 24h p.i. by TCID 50 infectivity assay. Data, expressed as TCID 50 /ml, represent the mean ± S.D. of duplicate samples. * = p < 0.05; ANOVA test. In parallel, cell viability (Δ) was determined in mock-infected cells by MTT assay. Absorbance (O.D.) of converted dye was measured at λ = 570nm. (E) Immunoblot of pHSF1-Ser326, HSF1, viral N and α-tubulin protein levels in samples treated as in D. (F) Western blot analysis of HSF1 protein levels in cytoplasmic (Cytosol) and nuclear (Nucleus) fractions of MRC-5 cells mock-infected (-) or infected (+) with HCoV-229E (0.1 TCID50/cell) for 24h and treated with DTHIB (10 μM, +) or vehicle (-) immediately after the adsorption period (left panel). 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 24h p.i. by TCID 50 infectivity assay (right panel). Data, expressed as TCID 50 /ml, represent the mean ± S.D. of duplicate samples. * = p < 0.05; Student’s t -test. (G) Confocal images of pHSF1-Ser326 (red) and α-tubulin (green) intracellular localization in MRC-5 cells mock-infected or infected with HCoV-229E (1 TCID50/cell) for 30h and treated with DTHIB (5 μM) or control diluent immediately after the adsorption period. Nuclei are stained with Hoechst (blue). Merge images are shown. Scale bar, 20 μm. (H) MRC-5 cells were mock-infected or infected with HCoV-229E (1 TCID50/cell) and treated with DTHIB (7,5 μM). At different times p.i., levels of pHSF1-Ser326, HSF1, HSP70, HSPA6, HSP60, AIRAP, viral N and α-tubulin proteins were determined by IB (top panels). In parallel, virus yield was determined by TCID50 infectivity assay (bottom panel). Data, expressed as TCID 50 /ml, represent the mean ± S.D. of duplicate samples. * = p < 0.05; Student’s t -test.

Article Snippet: Human normal lung MRC-5 fibroblasts (American Type Culture Collection, ATCC, CCL-171), Caco-2 hACE2 cells, stably expressing the hACE2 receptor, and HeLa cells, stably transfected with pSUPER-HSF1i/pcDNA (HeLa-HSF1i) or control (HeLa wild-type) plasmids, were grown at 37°C in a 5% CO 2 atmosphere in EMEM (MRC-5 cells) or DMEM (Caco-2 hACE2 and HeLa cells) medium, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.

Techniques: Western Blot, Transfection, Infection, Stable Transfection, Plasmid Preparation, Adsorption, MTT Assay, Staining

Journal: Plants

Article Title: Thymus zygis, Valuable Antimicrobial (In Vitro and In Situ) and Antibiofilm Agent with Potential Antiproliferative Effects

doi: 10.3390/plants12233920

Figure Lengend Snippet: The effects of TZEO on the viability of MRC-5 cells after 24 and 72 h of treatment. Results are presented as the mean of three independent experiments ± standard error: * p < 0.05 relative to control.

Article Snippet: To carry out this experiment, the human lung normal fibroblast cell line MRC-5, breast cancer cell line MDA-MB-231, colon cancer cell line HCT-116, and chronic myelogenous leukemia cell line K562 were obtained from the American Tissue Culture Collection.

Techniques: