Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of autophagy in the pathogenesis of exposure keratitis

doi: 10.1111/jcmm.14310

Figure Lengend Snippet: Autophagy induced by air‐lift injury is mTOR‐dependent, and is regulated by ER stress via the PI3K/Akt/mTOR pathway. A, Western blot showing the changes in p‐ULK1, p‐mTOR and mTOR expression in the HCECs cells treated with different air exposure times (6 h and 24 h), n = 3. B, Western blots with anti‐ p‐ULK1, p‐mTOR and mTOR antibodies in air‐lifted corneal epithelium with different exposure times (0 h, 0.5 h, 1 h, 2 h and 24 h), n = 3. (C, D) HCECs were incubated with DMSO or 10 μM salubrinal (Sal) for 6 h, and followed by air exposure for 6 h or 24 h. C, Western blot analysis shows that sal could restore air exposure increased p‐PERK, CHOP and LC3‐II and decreased p‐eIF2α, n = 3. D, Sal also restores air exposure‐induced phosphorylation of PI3K and Akt, n = 3

Article Snippet: The antibodies specific for PERK (3192), phospho‐PERK (Thr980, 3179), mTOR (2983), phospho‐mTOR (Sre2448, 2971), AKT (4691), phospho‐AKT (Sre473, 9271), phosphor‐ULK1 (Ser555, 5869) and CHOP (2895) were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Western Blot, Expressing, Incubation, Phospho-proteomics

Journal: Virulence

Article Title: Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

doi: 10.1080/21505594.2020.1845040

Figure Lengend Snippet: CSFV infection causes the PERK pathway-mediated autophagy to promote viral replication in cultured PK-15 cells . (a–d) PK-15 cells cultured in 12-well plates were respectively pretreated with 1 μM CCT, 1 μM GSK, 1 μM SAL or equal amount of DMSO for 1 h, and then subject to CSFV infection (1 MOI) for 1.5 h. The cells were further cultured in the presence of the chemicals for 24 h, and then collected for qRT-PCR (a), Western blot (b and c) and TCID 50 assays (d), respectively. For qRT-PCR (a), relative quantification of ATG5, Beclin1, ATF4, CHOP and NS5B genes were assessed using the 2 −ΔΔCT method and normalized to GAPDH . Two-way ANOVA tests: *, P < 0.05; **, P < 0.01. For Western blot (b), samples were prepared as described in the “materials and methods” for Western blot analyses using specific antibody against p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro and tubulin. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantification were performed with GraphPad Prism 6 software. Two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001 (c). For virus titration (d), the cells and supernatants of each treatment were collected for TCID 50 assays through an indirect immunofluorescence assay using mouse anti-CSFV E2 antibody. Virus titers were expressed as Log 10 (TCID 50 /mL). Two-way ANOVA tests: *, P < 0.05. (e–h) PK-15 cells cultured in 12-well plates were respectively transfected with 10 nM SiPERK or 10 nM SiNC for 4 ~ 6 h, and further incubated at 37°C for 12 h prior to a 1.5 h of incubation with 1 MOI of CSFV. The cells were further cultured for 24 h, and then collected for detection of relative mRNA expression of ATG5, ATG12, Beclin1 and NS5B genes by qRT-PCR (e), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro , and tubulin through Western blot analysis (f and g), as well as measurement of virus titers with TCID 50 assays (h). All the data shown are expressed as mean ± SD values of two independent experiments, and are analyzed by two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001

Article Snippet: The primary antibodies used in our study were specific for phosphor (p)-PERK (Thr980) (Cell Signaling, 3179), total PERK (Cell Signaling, 3192), p-eIF2α (S51) (Bioworld, BS4787), total eIF2α (Bioworld, BS3651), GRP78 (Santa Cruz, sc-13968), ATG5 (Novus Biologicals, NB110-53818), SQSTM1/p62 (Cell Signaling, 39749), LC3B (Cell Signaling, 2775), CSFV-E2 (MEDIAN/JBT, 9011), Tubulin (Beyotime, AT819), and CSFV-Npro (kindly donated by professor Xinglong Yu, Hunan Agricultural University, China).

Techniques: Infection, Cell Culture, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Software, Virus, Titration, Immunofluorescence, Transfection, Incubation, Expressing

Journal: Virulence

Article Title: Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

doi: 10.1080/21505594.2020.1845040

Figure Lengend Snippet: CSFV infection causes the PERK pathway-mediated autophagy to promote viral replication in cultured 3D4/2 cells . (a–d) 3D4/2 cells were treated with PERK pathway regulators and CSFV infection as described in the legend of , and then collected for detection of relative mRNA expression of ATG5, Beclin1, ATF4, CHOP and NS5B genes by qRT-PCR (a), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro , and tubulin through Western blot analysis (b and c), as well as measurement of virus titers with TCID 50 assays (d). (e–h) 3D4/2 cells were treated with SiPERK and CSFV infection as described in the legend of , and then collected for detection of relative mRNA expression of ATG5, ATG12, Beclin1 and NS5B genes by qRT-PCR (e), and determination of protein expression of p-PERK, total PERK, p-eIF2α, total eIF2α, ATG5, p62, LC3B, N pro and tubulin through Western blot analysis (f and g), as well as measurement of virus titers with TCID 50 assays (h). All the data shown are expressed as mean ± SD values of two independent experiments. Two-way ANOVA tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001

Article Snippet: The primary antibodies used in our study were specific for phosphor (p)-PERK (Thr980) (Cell Signaling, 3179), total PERK (Cell Signaling, 3192), p-eIF2α (S51) (Bioworld, BS4787), total eIF2α (Bioworld, BS3651), GRP78 (Santa Cruz, sc-13968), ATG5 (Novus Biologicals, NB110-53818), SQSTM1/p62 (Cell Signaling, 39749), LC3B (Cell Signaling, 2775), CSFV-E2 (MEDIAN/JBT, 9011), Tubulin (Beyotime, AT819), and CSFV-Npro (kindly donated by professor Xinglong Yu, Hunan Agricultural University, China).

Techniques: Infection, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Virus

Journal: Virulence

Article Title: Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

doi: 10.1080/21505594.2020.1845040

Figure Lengend Snippet: Proposed model for CSFV-induced autophagy via cellular PERK and IRE1 pathway . CSFV infection induces ERS and then triggers autophagy through the PERK-eIF2α-ATF4-CHOP and the IRE1/GRP78 pathway, thus facilitating viral replication in cultured cells

Article Snippet: The primary antibodies used in our study were specific for phosphor (p)-PERK (Thr980) (Cell Signaling, 3179), total PERK (Cell Signaling, 3192), p-eIF2α (S51) (Bioworld, BS4787), total eIF2α (Bioworld, BS3651), GRP78 (Santa Cruz, sc-13968), ATG5 (Novus Biologicals, NB110-53818), SQSTM1/p62 (Cell Signaling, 39749), LC3B (Cell Signaling, 2775), CSFV-E2 (MEDIAN/JBT, 9011), Tubulin (Beyotime, AT819), and CSFV-Npro (kindly donated by professor Xinglong Yu, Hunan Agricultural University, China).

Techniques: Infection, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Mutations in the selenocysteine insertion sequence-binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans

doi: 10.1172/JCI43653

Figure Lengend Snippet: (A) Anthropometric measures and body composition data in P1 and P2 are shown as z scores, relative to an age- and sex-matched normal reference population. (B) Quantitation of visceral (green) and subcutaneous (red) abdominal fat from cross-sectional magnetic resonance imaging scans in P1 and an age- and sex-matched control subject. (C–F) Abdominal visceral (C) and subcutaneous (D) fat in P1 (red symbol) and age- and sex-matched normal subjects (black symbols; n = 320) measured by ultrasound scan; circulating, fasting adiponectin (E) and insulin (F) levels in P1 (red symbol) and age- and sex-matched normal subjects (black symbols; n = 180). Normal individuals were subjects recruited to a local (MRC, Fenland) population-based cohort study. (G–I) Primary fibroblasts from P1 and an age- and sex-matched control subject were exposed to varying insulin concentrations. (G) Representative Western blot showing pAKT and pERK1/2 levels with calnexin as loading control. (H and I) Quantification of pERK1/2 and pAKT levels.

Article Snippet: Insulin signaling pathway Fibroblasts from P1 and control cells were serum deprived (16 hours), exposed to varying concentrations of insulin (5 minutes), lysed, and analyzed by Western blotting using specific antibodies as follows: pERK1/2 (T202/T204) (mouse monoclonal 9106, Cell Signalling); pAKT (S473) (rabbit monoclonal 9271, Cell Signalling); calnexin (rabbit polyclonal ab 13504, Abcam).

Techniques: Quantitation Assay, Magnetic Resonance Imaging, Western Blot