Journal: Nature Communications

Article Title: The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

doi: 10.1038/s41467-022-31181-y

Figure Lengend Snippet: a , b EMCV-Wt and EMCV-L Zn -infected HeLa cells and corresponding cell culture supernatants were harvested at the indicated timepoints after infection. Line graphs display the increase in intracellular virus titers ( a ) and virus titers in the cell culture supernatant ( b ) over time, as determined by end-point dilution. Depicted are mean values ± SD from n = 3 independent experiments. c 10 K and 100 K EVs were separated from naked virions using density gradient centrifugation and EV-enclosed virus titers were determined by end-point dilution assay. Bars display the mean fold infectivity of EMCV-L Zn EVs relative to EMCV-Wt EVs ± SD from n = 5 independent experiments. **** p < 0.0001, as assessed by a two-tailed one-sample t -test. ND not detected. d 100 K EVs released by cells infected with either EMCV-Wt or EMCV-L Zn were isolated and EV-protein composition was analyzed by LC-MS/MS. The number of unique EMCV peptides identified within and outside of the capsid-coding region are indicated. Source data are provided as a Source Data file.

Article Snippet: Membranes were incubated with blocking buffer (0.2% fish skin gelatin (FSG; Sigma-Aldrich) (CD9, flotillin, CD63, LC3) or 4% BSA (LC3, actin, EMCV 3D) + 0.1% Tween-20 in PBS) for 1 h, and for >16 h at 4 °C with the following primary antibodies: mouse-α-CD63 (1:1000, clone TS63; Abcam, Cambridge, UK), mouse-α-CD9 (1:2000, clone HI9a; Biolegend, San Diego, CA), mouse-α-Flotillin-1 (1:1000, clone 18/Flotillin-1; BD Biosciences), mouse-α-LC3 (1:500, clone 5F10; 1:50, clone 2G6; Nanotools, Teningen, Germany), rabbit-α-LC3 (1:1000, polyclonal; MBL International, Woburn, MA), mouse-α-actin (1:30,000, clone AC-15, Sigma-Aldrich), and mouse-α-EMCV 3D polymerase (1:100, clone 3B7, sc-65633, Santa Cruz Biotechnology, Dallas, TX) diluted in blocking buffer.

Techniques: Infection, Cell Culture, Virus, Gradient Centrifugation, End-point Dilution Assay, Two Tailed Test, Isolation, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

doi: 10.1038/s41467-022-31181-y

Figure Lengend Snippet: a 10,000× g (10 K) and 100,000× g (100 K) ultracentrifugation pellets were collected from an equal number of EV-producing cells 8 h after mock, EMCV-Wt, or EMCV-L Zn infection and analyzed by western blotting for the presence of EV-marker proteins CD9, CD63, and flotillin. Depicted is a representative of n = 3 independent experiments. b – g EVs were isolated from cell culture supernatants of mock, EMCV-Wt, and EMCV-L Zn infected cells by differential ultracentrifugation and density gradient centrifugation at indicated timepoints p.i. EVs were fluorescently labeled with CFSE and analyzed by high-resolution flow cytometry. b The contribution of 10 K vs. 100 K EVs to the overall EV release by mock, EMCV-Wt, and EMCV-L Zn infected cells was quantified. Mean values ± SD are presented for n = 3 independent experiments. **left p = 0.0025, **right p = 0.0015. c , d Depicted is the increase over time of 100 K EV numbers present in 1.06–1.10 g/ml fractions measured in a fixed time window of 30 s ( c ) and the number of EVs present in those density fractions at 8 h p.i. ( d ). ** p = 0.0047, * p = 0.0107. e Representative FSC/SSC dot plots are depicted of the samples in ( d ). Gates delineate FSC-high and FSC-low EVs. f , g Presented are the percentage of FSC-high EVs of the total EVs present in the 1.08 g/ml density fraction at the indicated timepoints ( f ) and the percentage of FSC-high EVs at 8 h p.i. ( g ). ** left p = 0.0061, ** right p = 0.0084. For ( c ) and ( f ) a representative image of n = 2 individual experiments is shown. For ( d ) and ( g ) mean values ± SD are presented for n = 4 independent experiments. p values were assessed by one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: Membranes were incubated with blocking buffer (0.2% fish skin gelatin (FSG; Sigma-Aldrich) (CD9, flotillin, CD63, LC3) or 4% BSA (LC3, actin, EMCV 3D) + 0.1% Tween-20 in PBS) for 1 h, and for >16 h at 4 °C with the following primary antibodies: mouse-α-CD63 (1:1000, clone TS63; Abcam, Cambridge, UK), mouse-α-CD9 (1:2000, clone HI9a; Biolegend, San Diego, CA), mouse-α-Flotillin-1 (1:1000, clone 18/Flotillin-1; BD Biosciences), mouse-α-LC3 (1:500, clone 5F10; 1:50, clone 2G6; Nanotools, Teningen, Germany), rabbit-α-LC3 (1:1000, polyclonal; MBL International, Woburn, MA), mouse-α-actin (1:30,000, clone AC-15, Sigma-Aldrich), and mouse-α-EMCV 3D polymerase (1:100, clone 3B7, sc-65633, Santa Cruz Biotechnology, Dallas, TX) diluted in blocking buffer.

Techniques: Infection, Western Blot, Marker, Isolation, Cell Culture, Gradient Centrifugation, Labeling, Flow Cytometry

Journal: Nature Communications

Article Title: The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

doi: 10.1038/s41467-022-31181-y

Figure Lengend Snippet: EVs released by cells infected with either EMCV-Wt or EMCV-L Zn were isolated and EV-protein composition was analyzed by LC-MS/MS. a Venn diagram depicting the number of unique and overlapping host proteins identified in at least 2 out of 3 replicate preparations of EMCV-Wt and EMCV-L Zn EVs. b – e Functional enrichment analysis for the 44 unique proteins in EMCV-Wt EVs was combined with an evaluation of protein–protein interactions using the STRING database. b , d Functional annotations are listed for the GO domains cellular component ( b ) and biological process ( d ). Bars depict the fold enrichment for the indicated GO-terms compared to the genome, with concomitant p values as assessed by one-tailed Fisher’s exact test with Bonferroni correction for multiple testing. c , e Proteins in interactome networks are indicated by their gene name and thickness of the connecting lines correlates with the strength of data support for interaction. Color-coding of individual proteins corresponds to their association with GO-terms that are enriched >10-fold compared to the genome for cellular component ( c ) or biological process ( e ). f Ultracentrifugation pellets (100 K) of cell culture supernatants from an equal number of mock, EMCV-Wt, or EMCV-L Zn infected cells were analyzed by western blotting for the presence of extracellular LC3. g EMCV-Wt samples from ( f ) were treated with proteinase K in the absence or presence of 0.2% triton and analyzed by western blotting for the presence of LC3. LC3I PC and LC3II PC are positive control samples for western blot detection of LC3I and LC3II, respectively. h EMCV-Wt samples from ( f ) were floated into a sucrose density gradient. Individual gradient fractions were analyzed for the presence of LC3 and the EV markers CD9 and flotillin by western blotting. Representative images of n = 3 independent experiments are shown for ( f , g ) and n = 2 independent experiments for ( h ). Source data are provided as a Source Data file.

Article Snippet: Membranes were incubated with blocking buffer (0.2% fish skin gelatin (FSG; Sigma-Aldrich) (CD9, flotillin, CD63, LC3) or 4% BSA (LC3, actin, EMCV 3D) + 0.1% Tween-20 in PBS) for 1 h, and for >16 h at 4 °C with the following primary antibodies: mouse-α-CD63 (1:1000, clone TS63; Abcam, Cambridge, UK), mouse-α-CD9 (1:2000, clone HI9a; Biolegend, San Diego, CA), mouse-α-Flotillin-1 (1:1000, clone 18/Flotillin-1; BD Biosciences), mouse-α-LC3 (1:500, clone 5F10; 1:50, clone 2G6; Nanotools, Teningen, Germany), rabbit-α-LC3 (1:1000, polyclonal; MBL International, Woburn, MA), mouse-α-actin (1:30,000, clone AC-15, Sigma-Aldrich), and mouse-α-EMCV 3D polymerase (1:100, clone 3B7, sc-65633, Santa Cruz Biotechnology, Dallas, TX) diluted in blocking buffer.

Techniques: Infection, Isolation, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Protein-Protein interactions, One-tailed Test, Cell Culture, Western Blot, Positive Control

Journal: Nature Communications

Article Title: The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

doi: 10.1038/s41467-022-31181-y

Figure Lengend Snippet: a Mock-infected cells and cells infected with EMCV-Wt or EMCV-L Zn were harvested 8 h p.i. Whole-cell lysates were analyzed for conversion of LC3I to LC3II by western blotting and the LC3II:LC3I ratios were calculated relative to mock-infected cells. *Indicates a detected LC3 band with intermediate size. Actin was included as a loading control and EMCV-3D to confirm infection. Depicted are representative blots and the mean ± SD of n = 4 independent experiments. * p = 0.0341 (bottom), p = 0.0257 (top) as determined by a two-tailed one-sample t -test. # p = 0.0437 using a two-tailed t -test. b Schematic overview of the mCherry-EGFP-LC3 autophagy reporter system. Using spot detection on LC3 reporter cells imaged by confocal microscopy, the accumulation of LC3 in autophagosomal vs. autolysosomal compartments can be monitored. Whereas autophagosomes appear as mCherry+ EGFP+ LC3+ puncta, autolysosomes are detected as mCherry+ EGFP− LC3+ puncta due to a loss of EGFP fluorescence in the acidic environment of the autolysosome lumen. c LC3 reporter cells were infected with EMCV-Wt or EMCV-L Zn for the indicated time or treated with bafA1 for 6 h and imaged for EGFP and mCherry fluorescence using confocal microscopy. Some EGFP+ compartments seem to lack mCherry signal, but this is likely due to the lower detection efficiency of mCherry versus EGFP. Bar graphs display a quantification of the ratio between the total surface area of EGFP and mCherry positive puncta as a measure for the ratio between the autophagosomes and autolysosomes present in the cells. Indicated are mean values ± SEM of ≥7 separate images with 2–5 cells each from a representative of n = 3 individual experiments. ** p = 0.0083, *** p = 0.0001, **** p < 0.0001 as assessed by one-way ANOVA with Dunnett’s multiple comparison test. Scale bar = 20 µm. ( d ) Infected cells were stained with 50 nM lysotracker and compared to mock-infected cells or bafA1 treated controls 6 h p.i. using live-cell confocal imaging. Depicted are representative images showing lysotracker fluorescence staining. Bar graphs display the average cellular staining intensity relative to uninfected controls. Indicated are mean values ± SD of five independent experiments. Scale bar = 30 µm. ** p = 0.0012, as assessed by a one-sample t -test. ns, p = 0.1400 as assessed by an unpaired, two-tailed t -test. Source data are provided as a Source Data file.

Article Snippet: Membranes were incubated with blocking buffer (0.2% fish skin gelatin (FSG; Sigma-Aldrich) (CD9, flotillin, CD63, LC3) or 4% BSA (LC3, actin, EMCV 3D) + 0.1% Tween-20 in PBS) for 1 h, and for >16 h at 4 °C with the following primary antibodies: mouse-α-CD63 (1:1000, clone TS63; Abcam, Cambridge, UK), mouse-α-CD9 (1:2000, clone HI9a; Biolegend, San Diego, CA), mouse-α-Flotillin-1 (1:1000, clone 18/Flotillin-1; BD Biosciences), mouse-α-LC3 (1:500, clone 5F10; 1:50, clone 2G6; Nanotools, Teningen, Germany), rabbit-α-LC3 (1:1000, polyclonal; MBL International, Woburn, MA), mouse-α-actin (1:30,000, clone AC-15, Sigma-Aldrich), and mouse-α-EMCV 3D polymerase (1:100, clone 3B7, sc-65633, Santa Cruz Biotechnology, Dallas, TX) diluted in blocking buffer.

Techniques: Infection, Western Blot, Control, Two Tailed Test, Confocal Microscopy, Fluorescence, Comparison, Staining, Imaging

Journal: Nature Communications

Article Title: The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

doi: 10.1038/s41467-022-31181-y

Figure Lengend Snippet: EMCV-Wt infected cells were treated with 200 nM rapamycin 1 h p.i. onwards to increase autophagic flux and enhance autolysosome formation. a Representative confocal microscopy images of infected mCherry-EGFP-LC3 reporter cells 6 h p.i. Bar graphs display the ratio between the total surface area of EGFP and mCherry positive puncta, that correspond to autophagosomes (EGFP+ mCherry+) and autolysosomes (EGFP−mCherry+). Indicated are mean values ± SEM of ≥7 separate images with 2–5 cells each from a representative of n = 3 individual experiments. *** p = 0.0008, * p = 0.0434 as assessed by one-way ANOVA with Tukey’s multiple comparison test. Scale bar = 10 μm. b Western blot analysis of LC3 release in ultracentrifugation pellets harvested 8 h p.i. from cell supernatants of an equal number of cells. Depicted is a representative image of n = 3 independent experiments. LC3I PC and LC3II PC are positive control samples for western blot detection of LC3I and LC3II, respectively. c , d 10 K and 100 K EVs were isolated and purified using density gradients. Depicted is the total amount of EV-associated infectivity ( c ) and its distribution over 100 K vs. 10 K EVs ( d ) (mean ± SD of n = 3 independent experiments) as determined by end-point dilution assay. *** p = 0.0009, ns p = 0.6599 as assessed by an unpaired, two-tailed t -test. Source data are provided as a Source Data file.

Article Snippet: Membranes were incubated with blocking buffer (0.2% fish skin gelatin (FSG; Sigma-Aldrich) (CD9, flotillin, CD63, LC3) or 4% BSA (LC3, actin, EMCV 3D) + 0.1% Tween-20 in PBS) for 1 h, and for >16 h at 4 °C with the following primary antibodies: mouse-α-CD63 (1:1000, clone TS63; Abcam, Cambridge, UK), mouse-α-CD9 (1:2000, clone HI9a; Biolegend, San Diego, CA), mouse-α-Flotillin-1 (1:1000, clone 18/Flotillin-1; BD Biosciences), mouse-α-LC3 (1:500, clone 5F10; 1:50, clone 2G6; Nanotools, Teningen, Germany), rabbit-α-LC3 (1:1000, polyclonal; MBL International, Woburn, MA), mouse-α-actin (1:30,000, clone AC-15, Sigma-Aldrich), and mouse-α-EMCV 3D polymerase (1:100, clone 3B7, sc-65633, Santa Cruz Biotechnology, Dallas, TX) diluted in blocking buffer.

Techniques: Infection, Confocal Microscopy, Comparison, Western Blot, Positive Control, Isolation, Purification, End-point Dilution Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

doi: 10.1038/s41467-022-31181-y

Figure Lengend Snippet: a EMCV-L Zn infected cells were treated with 500 nM apilimod for 16 h prior to infection or 200 nM BafA1 1 h p.i. onwards. EV-containing 100,000× g ultracentrifugation pellets were isolated from supernatants of an equal number of cells at 8 h p.i. and analyzed by western blot for the presence of extracellular released LC3. Depicted is a representative image of n = 2 independent experiments. LC3I PC and LC3II PC are positive control samples for western blot detection of LC3I and LC3II, respectively. b 100 K EVs were labeled with CFSE, purified by density gradient centrifugation, and analyzed by high-resolution flow cytometry. Bars indicate the mean fold increase in EV release relative to untreated controls ±SD from n = 3 (BafA1) or n = 4 (apilimod) independent experiments. ** p = 0.0064, ns (left) p = 0.1148 as assessed by a two-tailed one-sample t -test, or two-tailed t -test for ns (right) p = 0.9936. c , d Depicted is the mean fold increase in EV-enclosed infectivity ( c ) and intracellular virus titers ( d ) relative to untreated controls ±SD corresponding to the samples in ( b ) as determined by end-point dilution assay. For c * p = 0.0210, ns p = 0.1464, for d * p = 0.0285, ns p = 0.4226, as assessed by a two-tailed one-sample t -test. Source data are provided as a Source Data file.

Article Snippet: Membranes were incubated with blocking buffer (0.2% fish skin gelatin (FSG; Sigma-Aldrich) (CD9, flotillin, CD63, LC3) or 4% BSA (LC3, actin, EMCV 3D) + 0.1% Tween-20 in PBS) for 1 h, and for >16 h at 4 °C with the following primary antibodies: mouse-α-CD63 (1:1000, clone TS63; Abcam, Cambridge, UK), mouse-α-CD9 (1:2000, clone HI9a; Biolegend, San Diego, CA), mouse-α-Flotillin-1 (1:1000, clone 18/Flotillin-1; BD Biosciences), mouse-α-LC3 (1:500, clone 5F10; 1:50, clone 2G6; Nanotools, Teningen, Germany), rabbit-α-LC3 (1:1000, polyclonal; MBL International, Woburn, MA), mouse-α-actin (1:30,000, clone AC-15, Sigma-Aldrich), and mouse-α-EMCV 3D polymerase (1:100, clone 3B7, sc-65633, Santa Cruz Biotechnology, Dallas, TX) diluted in blocking buffer.

Techniques: Infection, Isolation, Western Blot, Positive Control, Labeling, Purification, Gradient Centrifugation, Flow Cytometry, Two Tailed Test, Virus, End-point Dilution Assay

Journal: Biomedicines

Article Title: Patient-Derived Cancer-Associated Fibroblasts Support the Colonization of Tumor Cells in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/biomedicines13020358

Figure Lengend Snippet: Holotomographic images of patient-derived CAF morphology in vitro. ( A ) An example of adherent–fibrotic, stellate-shaped morphology. ( B ) Intermediate CAFs with elongated and star-like cell morphology. ( C ) Invasive–migratory spindle-shaped morphology. Brighter regions correspond to areas of higher dry mass density. A 90 × 90 µm field of view is shown.

Article Snippet: Next, the cells were analyzed for their morphology and interaction under a 3D Cell Explorer-fluo holotomographic microscope (Nanolive SA, Tolochenaz, Switzerland) with an air objective at 60× magnification.

Techniques: Derivative Assay, In Vitro

Journal: Biomedicines

Article Title: Patient-Derived Cancer-Associated Fibroblasts Support the Colonization of Tumor Cells in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/biomedicines13020358

Figure Lengend Snippet: Holotomographic imaging of tumor cells with direct physical contact with CAFs. ( A ) Tumor cells containing lipid droplets (bright white dots) and vacuoles of different sizes (black circles). Fine adhesions can be seen attaching cells to the plate surface. ( B ) An example of a tumor cell with increased surface area, the appearance of stress fibers (straight-line structures), and further membrane disruption. ( C ) Tumor cells containing small vacuoles, seen as black circles (left). The disruption of tumor cell plasma membranes is also visible as brighter areas around the tumor cell perimeter. Brighter regions correspond to areas of higher dry mass density. A 90 × 90 µm field of view is shown.

Article Snippet: Next, the cells were analyzed for their morphology and interaction under a 3D Cell Explorer-fluo holotomographic microscope (Nanolive SA, Tolochenaz, Switzerland) with an air objective at 60× magnification.

Techniques: Imaging, Membrane, Disruption, Clinical Proteomics

Journal: Cell Death & Disease

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

doi: 10.1038/s41419-020-03046-z

Figure Lengend Snippet: a BMMs were cultured with RANKL (50 ng/mL) and various doses of thapsigargin (0, 0.05, 0.1, 0.2 nM) for 4 days, TRAP staining was conducted and TRAP-positive osteoclasts (≥3 nuclei) were counted. b , c RANKL (100 ng/mL) was used to stimulated BMMs differentiating into osteoclasts. Total proteins were extracted at different time points from 0 to 3 days of induction and western blot was performed to detect the proteins of PERK pathway. Densitometric analysis of an immunoblot from three independent experiments. * p < 0.05, ** p < 0.01 versus 0 day group. d – f Thapsigargin and GSK2606414 were added to osteoclast-induced differentiation medium containing RANKL (50 ng/mL), respectively. TRAP staining was performed 4 days later and osteoclasts were counted. Total protein was extracted on day 4 and detect the protein expression of p-PERK, PERK and osteoclast differentiation marker genes. Densitometric analysis of an immunoblot from three independent experiments. * p < 0.05, ** p < 0.01. TG represents thapsigargin in the figure.

Article Snippet: GSK2606414 and antioxidant N-acetylcysteine (NAC) were obtained from Selleck (Houston, United States).

Techniques: Cell Culture, Staining, Western Blot, Expressing, Marker

Journal: Cell Death & Disease

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

doi: 10.1038/s41419-020-03046-z

Figure Lengend Snippet: a – d BMMs were treated with PERK siRNA or small molecule inhibitor GSK2606414 (0.01, 0.05, 0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 4 days. Then, TRAP assay was performed. TRAP-positive multinucleated osteoclasts (≥3 nuclei) were counted. e – h BMMs were seeded in Osteo Aaasy Surface plate at a density of 2 × 10 4 cells/well, and cultured with 100 ng/mL RANKL and 30 ng/mL M-CSF. After mature osteoclasts were formed in each group, PERK siRNA and GSK2606414 were applied for 3 days. Bone resorption area was quantified by using Image J, Scale bar = 400 μm. Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Article Snippet: GSK2606414 and antioxidant N-acetylcysteine (NAC) were obtained from Selleck (Houston, United States).

Techniques: TRAP Assay, Cell Culture

Journal: Cell Death & Disease

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

doi: 10.1038/s41419-020-03046-z

Figure Lengend Snippet: BMMs were cultured with RANKL (100 ng/mL) and various concentrations of GSK2606414 (0.05, 0.1 nM) for 4 days, and actin ring fluorescence staining was performed. a , b F-actin ring fluorescence images were acquired and quantified. Scale bar = 400 μm, Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Article Snippet: GSK2606414 and antioxidant N-acetylcysteine (NAC) were obtained from Selleck (Houston, United States).

Techniques: Cell Culture, Fluorescence, Staining

Journal: Cell Death & Disease

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

doi: 10.1038/s41419-020-03046-z

Figure Lengend Snippet: a – c BMMs were treated with GSK2606414 (0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 2 or 4 days. p-PERK, PERK and osteoclast-related marker genes proteins expression were measured by western blot at the indicated times and mRNA levels were determined by RT-PCR. d – g BMMs were starved with a-MEM in the absence of FBS for 12 h, pretreated with or without GSK2606414 for 2 h. After that, BMMs were treated with or without RANKL (100 ng/mL) for the indicated times. The total proteins were extracted and western blot was conducted to detect the expression of NF-κB and MAPK signaling pathways. Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Article Snippet: GSK2606414 and antioxidant N-acetylcysteine (NAC) were obtained from Selleck (Houston, United States).

Techniques: Marker, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Protein-Protein interactions

Journal: Cell Death & Disease

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

doi: 10.1038/s41419-020-03046-z

Figure Lengend Snippet: a , b Micro-CT images of the distal femoral metaphyseal region from the SHAM, SHAM + GSK2606414, OVX, and OVX + GSK2606414 groups. Histograms represent the trabecular structural parameters of the distal femur: BV/TV, Tb.N, Tb.Th and Tb.Sp. Data are presented as means ± SEM, n = 8–10 mice per group. * p < 0.05 versus the OVX group. c , d TRAP and H&E staining were performed on sections of distal femurs. N.Oc/BS of the TRAP staining were calculated. Scale bar (40×) = 1000 μm, Scale bar (100×) = 400 μm. Data are presented as means ± SEM, n = 8–10 mice per group. ** p < 0.01 versus the OVX group.

Article Snippet: GSK2606414 and antioxidant N-acetylcysteine (NAC) were obtained from Selleck (Houston, United States).

Techniques: Micro-CT, Staining

Journal: Cell Death & Disease

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

doi: 10.1038/s41419-020-03046-z

Figure Lengend Snippet: a , b After BMMs were stimulated by RANKL (100 ng/mL) for 0, 1, 2 and 3 days, total proteins were extracted to detect the levels of Beclin1 and LC3B. * p < 0.05, ** p < 0.01 versus 0 day group. c , d Thapsigargin and GSK2606414 were added to osteoclast-induced differentiation medium containing RANKL (50 ng/mL), respectively. Total proteins were extracted on the 4th day and western blot was performed to examine the expression of autophagy-related proteins Beclin1 and LC3B. TG represents thapsigargin in the figure. e , f BMMs were treated with GSK2606414 (0.05, 0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 4 days, total proteins were extracted and western blot was used to verify the expression of Beclin1 and LC3B. ( g ) C57BL/6 mice were given GSK2606414 (50 mg/kg) by intragastric gavage every 2 days after ovariectomy. Six weeks later, femurs of mice were collected and sectioned, subjected to LC3 immunohistochemical staining. Scale bar = 400 μm, Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Article Snippet: GSK2606414 and antioxidant N-acetylcysteine (NAC) were obtained from Selleck (Houston, United States).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

doi: 10.1038/s41419-020-03046-z

Figure Lengend Snippet: a , b After infecting BMMs with mRFP-GFP-LC3 tandem fluorescent protein adenovirus, GSK2606414 and RANKL (100 ng/mL) were used to treat BMMs. On the 4th day, the fluorescence was observed with a confocal microscope and quantitative analysis was performed. Scale bar = 10 μm. c GSK2606414 and RANKL (100 ng/mL) were used to treat BMMs for 2 days, the formation of autophagosomes in BMMs was observed under transmission electron microscopy. Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Article Snippet: GSK2606414 and antioxidant N-acetylcysteine (NAC) were obtained from Selleck (Houston, United States).

Techniques: Fluorescence, Microscopy, Transmission Assay, Electron Microscopy

Journal: Cell Death & Disease

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

doi: 10.1038/s41419-020-03046-z

Figure Lengend Snippet: a BMMs were treated with GSK2606414 (0.1 μM) in the presence of RANKL (100 ng/mL) for 4 days, and DCFH-DA probe was used to detect intracellular ROS levels. Scale bar = 200 μm. b , c RANKL (100 ng/mL) and NAC were used to stimulate BMMs, after 4 days, the total proteins were extracted and western blot was conducted to test the expression of PERK and its phosphorylation level. d , e BMMs were treated with NAC and PERK activator CCT020312 in the presence of RANKL (100 ng/mL) for 4 days, total proteins were extracted and Western Blot was performed to examine the expression of osteoclast-related proteins NFATc1, c-fos and Autophagy-related proteins Beclin1, LC3B. f The schematic model of the hypothesized mechanism by which PERK inhibition affects osteoclast differentiation. Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Article Snippet: GSK2606414 and antioxidant N-acetylcysteine (NAC) were obtained from Selleck (Houston, United States).

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