Journal: American Journal of Translational Research

Article Title: CLOCK disruption aggravates carotid artery stenosis through endoplasmic reticulum stress-induced endothelial-mesenchymal transition

doi:

Figure Lengend Snippet: The ClockΔ19/Δ19 mutation exacerbated endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in vivo. A. p-PERK, p-eIF2α, p-IRE1α, XBP-1, and ATF6 protein levels in partially ligated CAs assayed by western blotting (n = 6). B. Immunofluorescence staining for CD31 (green) and XBP-1 (red). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. C. Immunocytochemical analysis of the percentage of XBP-1+ ECs in the intima of partially ligated CAs (n = 6). D. Immunofluorescence intensity of XBP-1 in the intima of partially ligated CAs (n = 6). *P < 0.05, **P < 0.01.

Article Snippet: Anti-ATF6 (ab37149) and anti-GAPDH (ab8245) antibodies were obtained from Abcam (Cambridge, MA, USA), anti-Xbp-1 (24385) was from Signalway Antibody (Baltimore, USA), anti-p-IRE1α (Ser724; {"type":"entrez-protein","attrs":{"text":"ARG40603","term_id":"1175807486","term_text":"ARG40603"}} ARG40603 ) and anti-p-eIF2α (Ser51; {"type":"entrez-protein","attrs":{"text":"ARG57722","term_id":"1176876255","term_text":"ARG57722"}} ARG57722 ) were from arigo Biolaboratories Corp (Taiwan), and anti-p-PERK (Thr980; 3179s) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Mutagenesis, In Vivo, Western Blot, Immunofluorescence, Staining

Journal: American Journal of Translational Research

Article Title: CLOCK disruption aggravates carotid artery stenosis through endoplasmic reticulum stress-induced endothelial-mesenchymal transition

doi:

Figure Lengend Snippet: ClockΔ19/Δ19 aggravated EndMT and endoplasmic reticulum (ER) stress induced by disturbed flow (DF) in vitro. A. Relative mRNA levels of Cdh5, S100a4, Acta2, Vimentin, Twist1, Snai1, Mmp2, and Mmp9 in pMAECs exposed to disturbed flow (DF) or undisturbed flow (UF) (n = 3). B, C. p-PERK, p-eIF2α, p-IRE1α, XBP-1, and ATF6 protein levels of pMAECs exposed to disturbed flow (DF) or undisturbed flow (UF) assayed by western blotting (n = 3). *P < 0.05 vs. WT+DF, **P < 0.01 vs. WT+DF, #P < 0.05 vs. WT+UF, ##P < 0.01 vs. WT+UF.

Article Snippet: Anti-ATF6 (ab37149) and anti-GAPDH (ab8245) antibodies were obtained from Abcam (Cambridge, MA, USA), anti-Xbp-1 (24385) was from Signalway Antibody (Baltimore, USA), anti-p-IRE1α (Ser724; {"type":"entrez-protein","attrs":{"text":"ARG40603","term_id":"1175807486","term_text":"ARG40603"}} ARG40603 ) and anti-p-eIF2α (Ser51; {"type":"entrez-protein","attrs":{"text":"ARG57722","term_id":"1176876255","term_text":"ARG57722"}} ARG57722 ) were from arigo Biolaboratories Corp (Taiwan), and anti-p-PERK (Thr980; 3179s) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vitro, Western Blot

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylation of eIF2α (S51), 4EBP1 (Ser65), S6K (Thr389), ULK1 (Ser757) and TFEB (Ser142) in U2OS cells treated with the indicated dose of LLOMe for 30 min. ( B ) Quantification by HCM of overlaps between mTOR and LAMP2 or G3BP1 puncta in U2OS cells. Cells were treated with EBSS, 2 mM LLOMe or 100 µM NaAsO2 for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified overlap between mTOR and LAMP2; red masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) and S6K1 (T389) in U2OS cells treated as in ( B ). ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) and cell death analysis by a LDH release assay in HEK293T cells expressing APEX2-eIF2α. Cells were treated with 1 mM LLOMe for the indicated durations. ( E ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells. Cells were treated with 2 mM LLOMe for the indicated durations. ( F ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with either scrambled siRNA as control (SCR) or MARK2 siRNA for knockdown (MARK2 KD ). Cells were treated with 2 mM LLOMe for 30 min. ( G ) Quantification by HCM of dsRNA puncta in U2OS cells. Cells were treated with 2 mM LLOMe or 100 ng/mL Poly (I:C) for 30 min. Green masks, computer-identified dsRNA puncta. ( H ) Immunoblot analysis of phosphorylation of PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or RNASET2 siRNA for knockdown (RNASET2 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. ( I ) Immunoblot analysis of phosphorylation of PKR (T446) in PKR KO U2OS G3BP1-GFP cells, overexpressing GFP, GFP-PKR and GFP-PKR K60A&K150A . Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. ( J ) Immunoblot analysis of phosphorylation of PKR (T446) in U2OS PACT knockdown cells (PACT KD ) overexpressing FLAG or FLAG-PACT. Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Figs. and .

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Western Blot, Lactate Dehydrogenase Assay, Expressing, Transfection, Control, Knockdown

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or eIF2α siRNA for knockdown (eIF2α KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of mTORC1 activity by phosphorylation of 4EBP1 (Ser65), S6K (Thr389), ULK1 (Ser757), and TFEB (Ser142) in U2OS cells transfected with either scrambled siRNA as control (SCR) or eIF2α siRNA for knockdown (eIF2α KD ). Cells were treated with 2 mM LLOMe for 30 min. Quantification is based on three independent experiments. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells overexpressing wild-type RagB (RagB WT ) or constitutively active RagB mutant (RagB Q99L ) treated with 2 mM LLOMe for 30 min. Quantification is based on three independent experiments. ( D ) Quantification by HCM of G3BP1 puncta in U2OS cells overexpressing wild-type RagB (RagB WT ) or constitutively active RagB mutant (RagB Q99L ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( E ) Quantification by HCM of G3BP1 puncta in eIF2α knockdown (eIF2α KD ) U2OS cells transfected with FLAG, FLAG- eIF2α WT or FLAG- eIF2α S51A . Cells were treated with 2 Mm LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( F ) Schematic summary of the findings in Figs. 2 and . NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Transfection, Control, Knockdown, Western Blot, Activity Assay, Mutagenesis

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantitative liquid chromatography-tandem mass spectrometry (LC/MS/MS) using the data-independent acquisition (DIA) technique to identify eIF2α binding partners that were proximity-biotinylated by APEX2-eIF2α during lysosomal damage (1 mM LLOMe for 1 h). Scatter (volcano) plot shows log2 fold change (LLOMe/CTR; spectral counts) and –log10 p value for the proteins identified and quantified in three independent experiments. Green dots indicate increase in proximity to eIF2α (log2 fold change ≥ 1), and red dots indicate decrease in proximity to eIF2α (log2 fold change ≤ −1) during LLOMe treatment. Orange dots indicate values below the statistical significance cut-off ( P ≥ 0.05). Bubble size represents a normalized value for the total amount of spectral counts for the protein indicated. PACT, PKR and ALIX proteins are highlighted as purple circles (see Dataset EV ). ( B ) Quantification by HCM of G3BP1-GFP puncta in wild type (WT) or PKR knockout (PKR KO ) U2OS G3BP1-GFP cells. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in WT or PKR KO U2OS G3BP1-GFP cells, as well as in cells overexpressing FLAG-PKR in PKR KO U2OS G3BP1-GFP cells. Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. ( D ) Co-IP analysis of interactions between eIF2α and PKR/PACT during lysosomal damage. HEK293T cells expressing FLAG (control) or FLAG-eIF2α were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or PACT siRNA for knockdown (PACT KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta; (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in SCR or PACT KD cells; 2 mM LLOMe for 30 min. The level of phosphorylation of PKR (T446) was quantified based on three independent experiments. ( F ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells treated with 1 mM LLOMe in the presence or absence of 210 nM imidazolo-oxindole C16 for 1 h. TMEM192-2xFLAG, control. The level of PKR, eIF2α and PACT in LysoIP was quantified based on three independent experiments shown in Fig. . ( G ) Schematic summary of the findings in Figs. 3 and . NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Knock-Out, Western Blot, Co-Immunoprecipitation Assay, Expressing, Control, Immunoprecipitation, Transfection, Knockdown, Purification

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Summary of the literature on the detected peptide count of PKR, PACT and eIF2α in the proteomic analysis of lysosomes based on LysoIP LC/MS/MS analysis. ( B ) Quantification of Fig. ; the level of PKR, eIF2α and PACT in LysoIP was quantified based on three independent experiments. ( C ) Confocal microscopy imaging of GFP-PKR and LAMP2 in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( D ) Confocal microscopy imaging of GFP-PACT and LAMP2 in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( E ) Confocal microscopy imaging of GFP-eIF2α and LAMP2 in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. * p < 0.05, ** p < 0.01, ANOVA. See also Fig. .

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Imaging

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells pre-treated with 15 µM BAPTA-AM for 1 h, subjected to 2 mM LLOMe treatment for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( G ) Schematic summary of the findings in Figs. 4 and . NT, untreated cells. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Transfection, Control, Knockdown, Western Blot

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of LAMP2 in U2OS cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). White masks, algorithm-defined cell boundaries; green masks, computer-identified LAMP2 puncta. ( B ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ), subjected to 2 mM LLOMe treatment for 30 min. ( D ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ), or pre-treated with 15 µM BAPTA-AM for 1 h. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALG2 knockdown cells (ALG2 KD ) overexpressing FLAG or FLAG-ALG2. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. .

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Transfection, Control, Knockdown, Western Blot

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or galectin-3 (Gal3) siRNA for knockdown (Gal3 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with scrambled siRNA as control (SCR), or galectin-3 (Gal3) siRNA for knockdown (Gal3 KD ), subjected to 2 mM LLOMe treatment for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Co-IP analysis of interactions among FLAG-Gal3, ALIX, PKR and PACT in HEK293T cells during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis for ALIX, PKR, and PACT based on three independent experiments. ( D ) Co-IP analysis of interactions between FLAG-PKR and PACT in HEK293T cells transfected with scrambled siRNA as control (SCR), or Gal3 siRNA for knockdown (Gal3 KD ) during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( E ) Co-IP analysis of interactions between Myc-PACT and PKR in HEK293T cells transfected with FLAG, or FLAG-Gal3 during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( F ) Co-IP analysis of interactions among FLAG-ALIX, PKR and PACT in HEK293T cells transfected with GFP, GFP-Gal3 or GFP-Gal3 R186S during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( G ) Schematic summary of the findings in Fig. 6. NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). ** p < 0.01, ANOVA. .

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Transfection, Control, Knockdown, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells infected with wild-type human adenovirus C2 (HAdV-C2 WT ) or C2 TS1 mutant (HAdV-C2 TS1 ) at MOI = 10 for 1 h. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells infected with wild type human adenovirus C2 (HAdV-C2 WT ) or C2 TS1 mutant (HAdV-C2 TS1 ) at MOI = 10 for 1 h. ( C ) Quantification by HCM of cell death by a propidium iodide (PI) uptake assay in U2OS wild type (WT) and G3BP1&2 double knockout (ΔΔG3BP1/2) cells during adenovirus infection. Cells were infected with wild-type human adenovirus C2 (HAdV-C2 WT ) at MOI = 10 for 1 h, and then stained with propidium iodide PI (dead cells) and Hoechst-33342 (total cells). White masks, algorithm-defined cell boundaries; red masks, computer-identified PI + nuclei. ( D ) Cell death analysis of supernatants of U2OS WT and ΔΔG3BP1/2 cells by a LDH release assay during SARS-Cov-2 ORF3a expression. Cells were transfected with the GFP-SARS-Cov-2 ORF3a construct overnight. ( E ) Cell death analysis of supernatants of human peripheral blood monocyte-derived macrophages (hMDM) by a LDH release assay during hemozoin exposure. Cells were treated with 10 µg/ml hemozoin for 4 h in the presence or absence of 1 μg/ml cycloheximide (CHX). ( F ) Quantification using AMNIS of cell death by Live/Dead TM stain kit in hMDM during silica treatment. Cells were treated with 200 µg/mL silica for 4 h in the presence or absence of 1 μg/ml cycloheximide (CHX), and then stained using Live/Dead TM stain kit (ThermoFisher). ( G ) Quantification using AMNIS of cell death by Live/Dead TM stain kit in hMDM during the treatment of tau oligomer. Cells were treated with 10 µg/mL tau oligomer for 4 h in the presence or absence of 1 μg/ml cycloheximide (CHX), and then stained using Live/Dead TM stain kit (ThermoFisher). CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). * p < 0.05, ** p < 0.01, ANOVA. See also Appendix Fig. S . .

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Infection, Mutagenesis, Western Blot, Double Knockout, Staining, Lactate Dehydrogenase Assay, Expressing, Transfection, Construct, Derivative Assay, Control

Journal: The EMBO Journal

Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

doi: 10.1038/s44318-024-00292-1

Figure Lengend Snippet: Reagents and tools table

Article Snippet: eIF2α gateway oligonucleotide sense 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGGGTCTAAGTTGTAGATTTTATC-3’ , GENEWIZ , N/A.

Techniques: Recombinant, Sequencing, Mutagenesis, Control, CRISPR, Staining, Magnetic Beads, Transfection, Lysis, Cytotoxicity Assay, Protease Inhibitor, Software

Journal: Microbiology Spectrum

Article Title: Quinoa Reduces High-Fat Diet-Induced Obesity in Mice via Potential Microbiota-Gut-Brain-Liver Interaction Mechanisms

doi: 10.1128/spectrum.00329-22

Figure Lengend Snippet: Quinoa may regulate TGR5, GLP-1, TLR4 and endoplasmic reticulum (ER) stress in the liver. (A) IHC of phosphorylated eukaryotic initiation factor 2 alpha (eIF-2α) in the liver. (B) IHC of TGR5 in the liver. (C) IHC of GLP-1 in the liver. (D) IHC of TLR4 in the liver. (E) Quantification AOD values of eIF-2α and TGR5 in the liver by Image J. (F) Quantification AOD values of GLP-1 and TLR4 in the liver by Image J. (G) Representative Western blot of the transcription factor C/EBP homologous protein (CHOP) and eIF-2α in the liver. (H) Quantitative assessment of the Western blot analysis results of CHOP and eIF-2α by Image J. *, P <0.05; **, P <0.01; compared with the control group and #, P <0.05; ##, P <0.01, versus the HFD group, determined by ANOVA and non-parametric test.

Article Snippet: IHC for GLP-1, claudin-1 in colons and brains as well as eIF-2α in livers were detected using the rabbit anti-GLP-1 (1:500; Servicebio), mouse anti-claudin-1 (1:500; Servicebio) and rabbit anti-eIF-2α (1:200; Servicebio) monoclonal antibodies.

Techniques: Western Blot