Journal: Cell & Bioscience

Article Title: Unraveling the protein kinase C/NDRG1 signaling network in breast cancer

doi: 10.1186/s13578-024-01336-z

Figure Lengend Snippet: NDRG1 is a stress-responsive protein. A Western blotting analysis for NDRG1 of lysates obtained from MDA-MB-231 cells exposed to 1% serum or maintained in a medium without lipids for 24 h. Cofilin was used as a loading control. B RT-qPCR of NDRG1 and NDRG3 mRNAs in control and MDA-MB-231 cells exposed to 1% serum for 24 h. The p-value was calculated using the Student’s t-test. The error bar represents ± SD. p-value *** < 0.001. C Western blotting analysis for NDRG1 of lysates obtained from MCF-7 and MDA-MB-231 cells exposed to 1% serum for 5 h or 24 h. Cofilin was used as a loading control. D Western blotting analysis for NDRG1, GRP78 and p62 of lysates obtained from MDA-MB-231 cells exposed to thapsigargin (1 μM) or LY294002 (10 μM) for 24 h. Cofilin was used as a loading control. E Representative images of MDA-MB-231 cells cultured in a normal condition medium or treated palimitic acid (200 μM), or oleic acid (200 μM) for 24 h. Images were acquired using an inverted wide-field microscope (EVOS FLoid Cell Imaging Station, Thermo). Scale bar 100 μm. F Western blotting analysis for NDRG1, GRP78 and p62 of lysates obtained from MDA-MB-231 cells exposed to palmitic acid or oleic acid at the concentration of 200 μM for 24 h. G Representative images of MDA-MB-231 cells cultured in a normal condition medium or treated with Vandetanib (10 μM), or Crizotinib (10 μM) for 24 h. Images were acquired using an inverted wide-field microscope (EVOS FLoid Cell Imaging Station, Thermo). Scale bar 100 μm. H Western blotting analysis for NDRG1, GRP78 and p62 of lysates obtained from MDA-MB-231 cells exposed to Vandetanib and Crizotinib treatment at the concentration of 10 μM for 24 h

Article Snippet: F Western blotting analysis for NDRG1, GRP78 and p62 of lysates obtained from MDA-MB-231 cells exposed to palmitic acid or oleic acid at the concentration of 200 μM for 24 h. G Representative images of MDA-MB-231 cells cultured in a normal condition medium or treated with Vandetanib (10 μM), or Crizotinib (10 μM) for 24 h. Images were acquired using an inverted wide-field microscope (EVOS FLoid Cell Imaging Station, Thermo).

Techniques: Western Blot, Control, Quantitative RT-PCR, Cell Culture, Microscopy, Imaging, Concentration Assay

Journal: Cell & Bioscience

Article Title: Unraveling the protein kinase C/NDRG1 signaling network in breast cancer

doi: 10.1186/s13578-024-01336-z

Figure Lengend Snippet: Different stress conditions activate PKC. Western blotting analysis for Phospho-PKC Substrate Motif [(R/K)XpSX(R/K)] of lysates obtained from MDA-MB-231 cells exposed to PMA, thapsigargin, Crizotinib, palmitic acid, and Vandetanib for 30 min

Article Snippet: F Western blotting analysis for NDRG1, GRP78 and p62 of lysates obtained from MDA-MB-231 cells exposed to palmitic acid or oleic acid at the concentration of 200 μM for 24 h. G Representative images of MDA-MB-231 cells cultured in a normal condition medium or treated with Vandetanib (10 μM), or Crizotinib (10 μM) for 24 h. Images were acquired using an inverted wide-field microscope (EVOS FLoid Cell Imaging Station, Thermo).

Techniques: Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation

doi: 10.1038/s41392-024-01836-x

Figure Lengend Snippet: Tyrosine kinase inhibitors targeting EGFR signalling pathway reduces SCoV2 propagation. a A scheme for primary screening of the antiviral effect of EGFR inhibitors against SCoV2 propagation. At 4 h post-infection, HEK293T cells infected with SCoV2 at an MOI of 1 were washed with fresh cell culture media 5 times and subsequently treated with the indicated EGFR inhibitors (10 μM) for 44 h. Cell culture supernatant and pellet were used for further analyses ( b , c ). b Extracellular RNA was isolated from culture supernatants of SCoV2-infected HEK293T cells and used for the quantification of SCoV2 RNA levels by real-time qRT-PCR. Data shown are the representative of two independent experiments (mean ± SD; n = 2). c Whole cell lysates of SCoV2-infected HEK293T cells were analysed by immunoblotting with antibody specific to SCoV2 N protein. β-actin, an internal loading control. d Rescue of SCoV2-induced mitochondrial translocation of EGFR by vandetanib treatment. Cytosolic (Cyto) and mitochondrial (Mito) fractions isolated from uninfected and SCoV2-infected HEK293T cells (MOI of 1) treated with vandetanib (10 μM) were analysed by immunoblotting with EGFR antibody. Fractions: purified cytoplasm, Cyto; purified mitochondria, Mito. Organelle marker: TOM20, mitochondria; GAPDH, cytoplasm. Infection marker: SCoV2 nucleocapsid (N). e Rescue of SCoV2-induced elevation in intracellular ATP level by vandetanib treatment. At 1 day post-treatment of vandetanib (10 μM), intracellular ATP level in SCoV2-infected HEK293T cells (MOI of 1) was analysed as described in Materials and Methods. Data shown are the average of two independent experiments (mean ± SD; n = 2; *p < 0.05). f , Western blot analysis of mitochondrial respiratory chain complex enzyme expression. At 1 day post-treatment of vandetanib (10 μM), the expression level of complex I, II, III, IV and V enzymes was analysed by immunoblotting with anti-Hu total OXPHOS complex antibody. C, complex; β-actin, an internal loading control; SCoV2 N, infection marker. g – h , HEK293T cells infected with SCoV2 for 4 h at an MOI of 1 were washed with fresh cell culture media 5 times and then further cultured for 20 h in the presence of vandetanib (10 μM) for RNAseq analysis. g Heat maps of relative mRNA expression of the indicated mitochondrial OXPHOS genes isolated from SCoV2-infected and vandetanib-treated SCoV2-infected cells. Each box indicates an average of three independent experiments. Colour indicates log2 fold-change for SCoV2-infected vs. uninfected cells and vandetanib-treated SCoV2-infected vs. SCoV2-infected cells, respectively. h Read coverage across the SCoV2 genome in the presence and absence of vandetanib. The graph represents the number of viral reads per position of the SCoV2 genome in HEK293T cells (SCoV2, dark blue; SCoV2/vandetanib, orange). A scaled model of the SCoV2 genome and its genes is portrayed below

Article Snippet: For the apoptosis analysis of SARS-CoV-2-infected cells induced by vandetanib treatment, an apoptosis assay was conducted using the Annexin V Apoptosis Detection Kit (eBioscience) following the manufacturer’s instructions.

Techniques: Infection, Cell Culture, Isolation, Quantitative RT-PCR, Western Blot, Control, Translocation Assay, Purification, Marker, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation

doi: 10.1038/s41392-024-01836-x

Figure Lengend Snippet: Vandetanib reveals a potent antiviral effect against various SCoV2 variants. a A scheme for analysing the antiviral effect of vandetanib (EGFR inhibitor) against various SCoV2 variants. HEK293T cells were infected with SCoV2 (S, V, G, GH and GR clades) and their variants of concern (VOC) including alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and omicron (B.1.529) variants, respectively, at an MOI of 1. At 4 h post-infection, HEK293T cells were washed with fresh cull culture media 5 times and then further incubated in the presence of vandetanib for 44 h. Cell culture media was used for further analyses of real-time qRT-PCR ( b ) and FFU assay using Vero E6 cells ( c ). b Real-time qRT-PCR data showing reduced extracellular SCoV2 RNA levels following treatment with vandetanib. Total RNA was isolated from the culture media of HEK293T cells infected with SCoV2 in the presence of vandetanib (10 μM) and then used for analysis of extracellular SCoV2 RNA level by real-time qRT-PCR using PCR primers set specific to the SCoV2 N gene. Data shown are the representative of two independent experiments (mean ± SD; n = 2). DMSO was used as the negative control. c FFU assay data showing the reduction in SCoV2 infectivity following treatment with vandetanib. Cell culture media of SCoV2-infected HEK293T cells post-treated with vandetanib at the indicated concentrations (1, 5 and 10 μM) were transferred to fresh Vero E6 cells and then further incubated for 8 h for FFU assay as described in Materials and Methods. SCoV2, cell culture media of SCoV2-infected HEK293T cells in the presence of vandetanib; Uninfected, cell culture media of SCoV2-uninfected HEK293T cells. The accompanying graphs show the average of two independent experiments (right panel). DMSO was used as the negative control. N.D. not determined

Article Snippet: For the apoptosis analysis of SARS-CoV-2-infected cells induced by vandetanib treatment, an apoptosis assay was conducted using the Annexin V Apoptosis Detection Kit (eBioscience) following the manufacturer’s instructions.

Techniques: Infection, Incubation, Cell Culture, Quantitative RT-PCR, Isolation, Negative Control

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation

doi: 10.1038/s41392-024-01836-x

Figure Lengend Snippet: Vandetanib is a potent antiviral agent for SCoV2 propagation. a Apoptosis analysis of SCoV2-infected and uninfected cells treated with vandetanib. HEK293T cells infected with SCoV2 at an MOI of 1 for 4 h were further cultured in the presence of vandetanib (1 or 10 μM) for 44 h. Apoptotic cell death was analysed by flow cytometry as described in Materials and Methods. Staurosporine (200 nM, 44 h) was used as a positive control for inducing apoptotic cell death. Data shown are the representative of two independent experiments. b Dose-dependent antiviral effect of vandetanib against SCoV2 infection. At 2 days post-treatment, whole cell lysates of SCoV2-infected HEK293T cells (MOI of 1) treated with vandetanib at the indicated concentrations were analysed by immunoblotting with SCoV2 N antibody. β-actin, an internal loading control. c Anti-SCoV2 effect by post-treatment of vandetanib. HEK293T cells infected with SCoV2 at an MOI of 1 for 4 h were washed with fresh cell culture media and then treated with vandetanib (0.1, 1, or 10 μM) for 44 h. d Anti-SCoV2 effect by pre-treatment of vandetanib. HEK293T cells were treated with vandetanib (0.1, 1, or 10 μM) for 4 h and then infected with SCoV2 at an MOI of 1 for 44 h. Intracellular SCoV2 RNA level in SCoV2-infected cells was analysed by real-time qRT-PCR using PCR primers set specific to the SCoV2 N gene ( c , d ). Data shown are the representative of two independent experiments (mean ± SD; n = 2). e – h , In vivo anti-SCoV2 efficacy of vandetanib in hACE2 transgenic mouse model susceptible to SCoV2 infection. e A scheme for analysing clinical disease score and inflammation in SCoV-2-infected mice orally administrated with vandetanib ( f , g ). Eight-week-old hACE2 transgenic mice (n = 5 per group) were intranasally (IN) inoculated with SCoV2 (2 × 10 3 pfu/head, 10 MLD 50 , clade S). One hour later, they were orally administrated with vandetanib (25 mg/kg) daily. At 6 days post-infection, all mice were terminated for further analyses ( f , g ). f Clinical scores of SCoV2-uninfected hACE2 transgenic mice (open circle), SCoV2-infected hACE2 transgenic mice administrated with vehicle (black circle) and SCoV2-infected hACE2 transgenic mice administrated with vandetanib (red circle). Vehicle control, PBS with 1% Tween 80 ( f – h ). Clinical scores for all mice were monitored daily based on ruffled fur (1 point), reduced mobility (1 point), hunched posture (1 point) and death (4 points) as described in Materials and Methods. g Immunohistochemistry analysis showing the effect of vandetanib in SCoV2-induced severe lung injury in hACE2 transgenic mouse. At 6 days post-infection, immunohistochemistry analysis was performed as described in Materials and Methods. Black scale bar, 500 μm (upper), 100 μm (lower). H&E score (right panel, mean ± SD; n = 12; * p < 0.0001). h In vivo anti-SCoV2 efficacy of vandetanib in hACE2 transgenic mice. Eight-week-old hACE2 transgenic mice (n = 3 per group) were intranasally (IN) inoculated with SCoV2 (2 × 10 3 pfu/head, clade S). One hour later, they were orally administrated with vandetanib (25 mg/kg) daily. At 3 days post-infection, all mice were terminated for further analyses (upper panel). Intracellular SCoV2 RNA levels of lung tissues isolated from SCoV2-infected hACE2 transgenic mice was analysed by real-time qRT-PCR. Each data point represents the average of two independent experiments (mean ± SD; n = 2, lower panel)

Article Snippet: For the apoptosis analysis of SARS-CoV-2-infected cells induced by vandetanib treatment, an apoptosis assay was conducted using the Annexin V Apoptosis Detection Kit (eBioscience) following the manufacturer’s instructions.

Techniques: Infection, Cell Culture, Flow Cytometry, Positive Control, Western Blot, Control, Quantitative RT-PCR, In Vivo, Transgenic Assay, Immunohistochemistry, Isolation