Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) The bar graphs show relative expression levels of CD44 in the indicated samples. (B–D) Maximum lifespan is plotted against CD44 expression levels in (B) cultured fibroblasts, (C) liver, and (D) skin. Results of Pearson and Spearman correlation tests are shown on the plots.

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Expressing, Cell Culture

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) A representative immunoblot of CD44 in mouse and NMR OPCs. (B) A representative immunoblot of CD44 in NMR OPCs transfected with siLuciferase, siCD44–1, or siCD44–2. Cells were collected 3 days after transfection. (C) The heatmap shows the relative levels of genes that are differently expressed between control and CD44-knockdown NMR OPCs (q value <0.05). (D) The boxplot shows the effects of CD44 knockdown on the expression levels of the indicated gene sets in NMR OPCs. The effects are shown for whole genes, the genes that were differentially expressed in NMR OPCs compared with OPCs of all other species, and the genes that were differentially expressed in both NMR and human OPCs compared with mouse and rat OPCs. (E) Functional enrichment analyses of genes that were differentially expressed between control and CD44 knockdown NMR OPCs. (F) The results of transcription factor target enrichment analysis of genes that were downregulated by CD44 knockdown in NMR OPCs. Top five overrepresented transcription factors obtained by the search of ARCHS4 TFs. Co-expression database and TRRUST TF-target regulatory interaction database are shown. (G) The heatmap shows the relative expression levels of differentially expressed genes that are associated with the terms highlighted in (E). (H) A representative immunoblot of CD44 in mouse OPCs transfected with siLuciferase, siCD44–1, or siCD44–2. Cells were collected 3 days after transfection. (I) Cell survival rate of mouse and NMR OPCs transfected with siLuc, siCD44–1, and siCD44–2 after 6 days of 0.5 μg/mL tunicamycin treatment. Transfection was performed 2 days before starting tunicamycin treatment (n = 8). (J) A representative immunoblot of CD44 in mouse OPCs. Cells were transfected with empty vector or vector encoding mouse CD44, NMR CD44, or mouse LDLR 2 days before sample collection. (K) Cell survival rate of mouse OPCs after 6 days of 0.5 μg/mL tunicamycin treatment. Cells were transfected with empty vector or vector encoding mouse CD44, NMR CD44, or mouse LDLR 1 day before starting tunicamycin treatment (n = 8). Error bars are presented as mean ± SD values. *p < 0.05; Wilcoxon test for (D), one-way ANOVA with post hoc Sidak’s test for (I), and one-way ANOVA with post hoc Dunnett’s test for (K).

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Western Blot, Transfection, Control, Knockdown, Expressing, Functional Assay, Plasmid Preparation

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) A representative immunoblot of CD44 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. (B) Cell survival rate of shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment (n = 10). (C) Functional enrichment analyses of genes that were up- or downregulated (q value <0.05) by both shCD44–1 and shCD44–2 expression in IMR90-hTert cells. (D) Real-time qPCR data of representative UPR genes in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells (n = 3). Data were normalized to GAPDH . (E) A representative immunoblot of HSPA5 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. (F) Relative density of HSPA5/b-Actin analyzed by ImageJ (n = 3). (G) The boxplot shows the effects of CD44 overexpression on the expression levels of the indicated genes in IMR90 cells. The effects are shown for the genes that are upregulated by ATF6 activating molecule AA147 and the genes co-expressed with ATF6, ATF4, or XBP1 (retrieved from Enrichr). (H) Percentages of overlap between CD44 co-expressing genes (retrieved from COXPRESdb v7) and the indicated genes. (I) Relative cell survival rate of shCD44–1 and shCD44–2 IMR90-hTert cells compared with shLuc IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment. Vehicle (DMSO) or 10 μM HA15 was added to the medium during tunicamycin treatment (n = 10). All immunoblots were repeated once with similar results. Error bars are presented as mean ± SD values. *p < 0.05; one-way ANOVA with post hoc Dunnett’s test for (B and F), two-way ANOVA with post hoc Dunnett’s test for (D), Wilcoxon test for (G), Fisher’s exact test for (H), and two-tailed t test with Bonferroni-Dunn correction for (I).

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Western Blot, Functional Assay, Expressing, Over Expression, Two Tailed Test

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) Protein-protein interactions among the 41 CD44-associated proteins plus IRE1, PERK, and ATF6 were mapped using the STRING software. Only highest confidence interactions (interaction score ≥0.9) that are experimentally determined (purple line) or stored in curated databases (blue line) were included in the analysis. IRE1, PERK, and ATF6 are shown as red nodes and the 16 CD44-associated ER proteins are shown as blue nodes. CD44-associated proteins are defined here as proteins that are detected by cross-linked immunoprecipitation-mass spectrometry of IMR90 cells using anti-CD44 antibody with at least 10-fold greater abundance than in control experiment using normal rabbit IgG antibody. (B) Representative immunoblots of CALR, HSP47, and CD44 in whole cell lysate and in CD44-immunoprecipitates prepared from IMR90 cells. The experiment was repeated once with similar results. (C) Representative confocal images of IMR90 cells co-stained with antibodies against CD44 and organelle markers (mitochondrial marker mtTFA and ER markers ERp57, HSP47, and RPN2). (D) The dot plot shows Pearson correlation coefficients between immunofluorescence signals of CD44 and organelle markers. Each dot represents a single cell (n = 30). Error bars are presented as mean ± SD values. Scale bars, 10 μm.

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Protein-Protein interactions, Software, Immunoprecipitation, Mass Spectrometry, Control, Western Blot, Staining, Marker, Immunofluorescence

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) The heatmap shows relative abundances of ER-associated proteins detected by mass spectrometry of ER fractions isolated from shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Cells were collected 24 h after starting 0.5 μg/mL tunicamycin treatment. ER-associated proteins were defined here as the proteins whose ER localization have been confirmed by the Human Protein Atlas or associated with the GO term “endoplasmic reticulum lumen.” (B) Representative immunoblots of COL6A3, SEC62, and HSPA5 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Whole cell lysates and culture supernatants were collected 24 h after starting 0.5 μg/mL tunicamycin treatment. (C) Representative immunoblots of ATF6, IRE1, PERK, CALR, EGFR, and Vinculin in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Cells were lysed in buffer containing either 1% Triton or 2% SDS. (D) A representative immunoblot of ATF6 in U2OS cells and in ATF6 KO U2OS cells infected with empty vector or vector expressing wild-type ATF6, ATF6 Y392C, or ATF6 Y567N. (E) Cell survival rate of ATF6 KO U2OS cells after 5 days of 2 μg/mL tunicamycin treatment (n = 8). Cells were infected with vector expressing wild-type ATF6, ATF6 Y392C, or ATF6 Y567N and selected with 100 μg/mL hygromycin prior to the experiment. (F) Relative expression levels of HSP90B1 and HSP5A in CD44 KO U2OS cells (n = 3). Cells were infected with empty vector or vector encoding CD44-ectodomain-KDEL 3 days before sample collection. Data were normalized to GAPDH . (G) Cell survival rate of CD44 KO U2OS cells after 5 days of 2 μg/mL tunicamycin treatment. Cells were infected with empty vector or vector encoding CD44-ectodomain-KDEL 3 days before starting tunicamycin treatment (n = 8). All immunoblots were repeated at least once with similar results. Error bars are presented as mean ± SD values. *p < 0.05; two-way ANOVA with post hoc Dunnett’s test (E) and two-tailed t test with (F) or without (G) Bonferroni-Dunn correction.

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Mass Spectrometry, Isolation, Western Blot, Infection, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-CD44 antibody , Proteintech , Cat#15675–1-AP; RRID:AB_2919823.

Techniques: Recombinant, Transfection, Protease Inhibitor, CyQUANT Assay, Proliferation Assay, Expressing, Mass Spectrometry, Software

Journal: Scientific Reports

Article Title: MiR-21-5p Links Epithelial-Mesenchymal Transition Phenotype with Stem-Like Cell Signatures via AKT Signaling in Keloid Keratinocytes

doi: 10.1038/srep28281

Figure Lengend Snippet: After keloid keratinocytes and normal skin keratinocytes were transfected with miR-21-5p mimic or inhibitor and their corresponding negative controls for 72 or 48 hours, respectively, ( a,b ) the average sphere numbers and cells number were detected by the sphere-forming assay. ( c ) The expressions of stem-like cells-associated markers (CD44 and ALDH1) were examined by western blot analysis and normalized by the expression of GAPDH. The western blot data are expressed as fold change relative to the mean value of control group. Statistical significance was obtained using Independent-Samples t-test for sphere-forming assay and Paired t-test for western blot analysis. Data was shown as the mean ± SEM of 3 independent cells from 3 replicates. Scale bar: 100 μm. (*p < 0.05).

Article Snippet: The specific primary antibodies against E-cadherin, p-ERK (rabbit polyclonal, 1:100, Santa Cruz Biotechnology), and PTEN, vimentin (mouse monoclonal, 1:100, Santa Cruz Biotechnology), CD44 (rabbit polyclonal, 1:2000, Proteintech Group, Chicago, IL), ALDH1 (rabbit polyclonal, 1:2000, GeneTex, Irvine, CA), ERK (rabbit polyclonal, 1:200, GeneTex), p-Akt and Akt (rabbit polyclonal, 1:1000, Cell Signaling Technology, Beverly, MA), and horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:4000, Santa Cruz Biotechnology) were used in western blot analysis.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: MiR-21-5p Links Epithelial-Mesenchymal Transition Phenotype with Stem-Like Cell Signatures via AKT Signaling in Keloid Keratinocytes

doi: 10.1038/srep28281

Figure Lengend Snippet: After keloid keratinocytes were transfected with miR-21-5p mimic and negative mimic control for 72 hours or miR-21-5p inhibitor and the negative inhibitor control for 48 hours, ( a ) immunofluorescence dual staining was used to detect the expression of CD44 (positive green staining) and vimentin (positive red staining) in keloid keratinocytes. Nuclei were visualized by staining with DAPI (blue). Scale bars: 50 μm. ( b ) The expression of PTEN, p-AKT and p-ERK were tested by western blot analysis, and were normalized by the expression of GAPDH and presented as fold change relative to the mean value of control group. Statistical significance was obtained using Paired t-test for western blot analysis. Data was shown as the mean ± SEM of 3 independent cells from 3 replicates. (*p < 0.05, **p < 0.01).

Article Snippet: The specific primary antibodies against E-cadherin, p-ERK (rabbit polyclonal, 1:100, Santa Cruz Biotechnology), and PTEN, vimentin (mouse monoclonal, 1:100, Santa Cruz Biotechnology), CD44 (rabbit polyclonal, 1:2000, Proteintech Group, Chicago, IL), ALDH1 (rabbit polyclonal, 1:2000, GeneTex, Irvine, CA), ERK (rabbit polyclonal, 1:200, GeneTex), p-Akt and Akt (rabbit polyclonal, 1:1000, Cell Signaling Technology, Beverly, MA), and horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:4000, Santa Cruz Biotechnology) were used in western blot analysis.

Techniques: Transfection, Control, Immunofluorescence, Staining, Expressing, Western Blot

Journal: Autophagy

Article Title: Burkholderia pseudomallei interferes with host lipid metabolism via NR1D2 -mediated PNPLA2/ATGL suppression to block autophagy-dependent inhibition of infection

doi: 10.1080/15548627.2020.1801270

Figure Lengend Snippet: Inhibition of PNPLA2 contributes to lipid accumulation after B. pseudomallei infection. (A) Expression of lipid metabolism-related genes in A549 cells infected with B. pseudomallei (MOI = 10) for 12 h. The mRNA levels of FASN, MGAT1, MGAT2, MGAT4C, MGAT5, DGAT1, DGAT2, PNPLA2, LIPE and MGLL were measured by qRT-PCR. (B and C) Western blot analysis of PNPLA2 and FASN in A549 cells after B. pseudomallei infection (MOI = 10) for 6, 12 and 24 h or at indicated MOIs (1, 10, 50, 100) for 12 h. Densitometric analyses were performed to quantitate the fraction of PNPLA2 of at least three independent experiments. (D) qRT-PCR analysis of PNPLA2 expression in BEAS-2B, H460 and H358 cells after treated with B. pseudomallei (MOI = 10) for 12 h. (E) Measurement of PNPLA2 expression by qRT-PCR in A549 cells after infected with B. thailandensis, S . Typhimurium and E. coli (MOI = 10) for 12 h, respectively. (F) The protein level of PNPLA2 was determined by western blot. A549 cells were transfected with the pcDNA3.1 or pcDNA3.1- PNPLA2 for 24 h. (G) Lipid droplets were detected by confocal microscopy. A549 cells were transfected with pcDNA3.1 or pcDNA3.1- PNPLA2 for 24 h, and then infected with B. pseudomallei (MOI = 10) for 12 h. Scale bars: 10 μm. (H and I) The numbers of lipid droplet puncta and B. pseudomallei in each cell were counted. Lipid droplets were staining by BODIPY (red). (J) Cellular lipid levels were detected by LC-MS/MS analysis. A549 cells were transfected with pcDNA3.1- PNPLA2 plasmids for 24 h, then uninfected or B. pseudomallei -infected (MOI = 10) for 12 h. This experiment was repeated at least three times. *P < 0.05, **P < 0.01. ns, not significant

Article Snippet: Cells were transfected with ATG7 siRNA (sc-41447), NR1D2 shRNA (sc-61456-SH) and PNPLA2 shRNA (sc-60223-SH), which were purchased from Santa Cruz Biotechnology, using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) for 24 h. The effects of ATG7 siRNA, NR1D2 shRNA and PNPLA2 shRNA were compared with those of a nontargeting control siRNA or control shRNA.

Techniques: Inhibition, Infection, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Confocal Microscopy, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Autophagy

Article Title: Burkholderia pseudomallei interferes with host lipid metabolism via NR1D2 -mediated PNPLA2/ATGL suppression to block autophagy-dependent inhibition of infection

doi: 10.1080/15548627.2020.1801270

Figure Lengend Snippet: PNPLA2 and lipophagy regulate the lipid accumulation during B. pseudomallei infection. (A) Representative images show the number of lipid droplets in A549 cells infected with B. pseudomallei (MOI = 10) for 12 h in the presence of Rapa (200 nM) or 3-MA (10 mM). Scale bars: 10 μm. (B and C) ImageJ quantification of the numbers of lipid droplets and B. pseudomallei per cell in above confocal images. (D) Lipophagy is involved in the regulation of TG in A549 cells. After pretreatment by DMSO, Rapa and 3-MA, or transfected with ATG7 siRNA (100 nM), cells were infected with B. pseudomallei (MOI = 10) for 12 h. (E) Lipid droplet was visualized by immunostaining in A549 cells. After transfected with control or PNPLA2 shRNA (1 μg), cells were infected with B. pseudomallei (MOI = 10) for 12 h in the presence of DMSO or Rapa (200 nM). Scale bars: 5 μm. (F and G) Quantification of the numbers of Nile red puncta and B. pseudomalle in each cell. (H and I) Measurement of TG turnover in A549 cells. After transfected with the pcDNA3.1 or pcDNA3.1- PNPLA2 , cells were treated with 3-MA or ATG7 siRNA and then infected with B. pseudomallei for 12 h. Experiments performed in triplicate showed consistent results. *P < 0.05, **P < 0.01. ns, not significant

Article Snippet: Cells were transfected with ATG7 siRNA (sc-41447), NR1D2 shRNA (sc-61456-SH) and PNPLA2 shRNA (sc-60223-SH), which were purchased from Santa Cruz Biotechnology, using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) for 24 h. The effects of ATG7 siRNA, NR1D2 shRNA and PNPLA2 shRNA were compared with those of a nontargeting control siRNA or control shRNA.

Techniques: Infection, Transfection, Immunostaining, Control, shRNA

Journal: Autophagy

Article Title: Burkholderia pseudomallei interferes with host lipid metabolism via NR1D2 -mediated PNPLA2/ATGL suppression to block autophagy-dependent inhibition of infection

doi: 10.1080/15548627.2020.1801270

Figure Lengend Snippet: PNPLA2 overexpression promotes autophagic flux and restricts B. pseudomallei replication. (A) PNPLA2 overexpression increased the LC3B-II:LC3B-I ratio and LAMP1 protein levels and decreased SQSTM1 expression as analyzed via western blot. A549 cells were transiently transfected with the pcDNA3.1 or pcDNA3.1- PNPLA2 plasmids for 24 h and then infected with B. pseudomallei for 12 h (MOI = 10). (B) PNPLA2 overexpression increased lysosomal association with GFP-LC3B in A549 cells infected with B. pseudomallei (MOI = 10) for 12 h. Scale bars: 10 μm. (C and D) Quantitative analysis of GFP-LC3B associated with LysoTracker and B. pseudomallei numbers per cell in (B). (E) A549 cells transiently expressing mRFP-GFP-LC3B by adenoviruses transduction were transfected with control or pcDNA3.1- PNPLA2 plasmids, and then infected with B. pseudomallei (MOI = 10) for 12 h. Scale bars: 10 μm. (F and G) The graph shows the quantification of mRFP + GFP + or mRFP + GFP − by taking the average number of puncta and B. pseudomallei numbers per cell in (E). (H) Representative images show lipid droplets by immunostaining. After transfected with the pcDNA3.1 or pcDNA3.1- PNPLA2 , A549 cells were treated with ATG7 siRNA or control and then infected with B. pseudomallei (MOI = 10) for 12 h. Scale bars: 5 μm. (I and J) Quantification of the numbers of BODIPY puncta and B. pseudomallei in each cell in (H). (K and L) Intracellular growth of B. pseudomallei was detected by CFU assay. Cells were treated as described in (H). Data is shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. ns, not significant

Article Snippet: Cells were transfected with ATG7 siRNA (sc-41447), NR1D2 shRNA (sc-61456-SH) and PNPLA2 shRNA (sc-60223-SH), which were purchased from Santa Cruz Biotechnology, using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) for 24 h. The effects of ATG7 siRNA, NR1D2 shRNA and PNPLA2 shRNA were compared with those of a nontargeting control siRNA or control shRNA.

Techniques: Over Expression, Expressing, Western Blot, Transfection, Infection, Transduction, Control, Immunostaining, Colony-forming Unit Assay

Journal: Autophagy

Article Title: Burkholderia pseudomallei interferes with host lipid metabolism via NR1D2 -mediated PNPLA2/ATGL suppression to block autophagy-dependent inhibition of infection

doi: 10.1080/15548627.2020.1801270

Figure Lengend Snippet: NR1D2 mediates lipid accumulation through inhibiting PNPLA2 expression following B. pseudomallei infection. (A and B) The mRNA and protein levels of NR1D2 were measured in A549 cells after transfection with control or NR1D2 shRNA (1 μg) for 24 h. (C and D) The mRNA and protein levels of PNPLA2 in control and NR1D2 -silenced A549 cells after infected with B. pseudomallei (MOI = 10) for 12 h. (E) The expression of PNPLA2 was measured in A549 cells. After pretreatment with SR9009 or GSK4112 in different concentrations (10 μM and 20 μM) for 48 h, cells were infected with B. pseudomallei (MOI = 10) for 12 h. (F) A549 cells were transfected with either pGL3-Basic empty vector (200 ng) or −2000/+20-LUC PNPLA2 construct (200 ng) plus pRL-TK-Renilla (10 ng) and then infected with B. pseudomallei (MOI = 10) for 12 h. Luciferase activities were measured and normalized to Renilla internal control. (G) −2000/+20 LUC PNPLA2 (200 ng) plus pRL-TK-Renilla (10 ng) was transfected into A549 cells with control or pcDNA3.1- NR1D2 (200 ng) for 24 h. (H) Representative images of control and NR1D2 -silenced A549 cells infected with B. pseudomallei for 12 h. Scale bar: 10 μm. (I and J) Quantitative analysis of the number of lipid droplets and the colocalization of PNPLA2 and BODIPY shown in (H). Data is shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01

Article Snippet: Cells were transfected with ATG7 siRNA (sc-41447), NR1D2 shRNA (sc-61456-SH) and PNPLA2 shRNA (sc-60223-SH), which were purchased from Santa Cruz Biotechnology, using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) for 24 h. The effects of ATG7 siRNA, NR1D2 shRNA and PNPLA2 shRNA were compared with those of a nontargeting control siRNA or control shRNA.

Techniques: Expressing, Infection, Transfection, Control, shRNA, Plasmid Preparation, Construct, Luciferase

Journal: Autophagy

Article Title: Burkholderia pseudomallei interferes with host lipid metabolism via NR1D2 -mediated PNPLA2/ATGL suppression to block autophagy-dependent inhibition of infection

doi: 10.1080/15548627.2020.1801270

Figure Lengend Snippet: NR1D2 is required for PNPLA2 -mediated effects on autophagic flux and regulation of B. pseudomallei infection. (A and B) Representative images of lipid droplet and GFP-LC3B puncta in A549 cells. After transfected with the control and NR1D2 shRNA, A549 cells were treated with GFP-LC3B and then infected with B. pseudomallei (MOI = 10) for 12 h. Quantitative analysis of the colocalization of BODIPY and GFP-LC3B. Scale bars: 5 μm. (C) The autophagic flux was detected by western blot. A549 cells were transiently transfected with the control and NR1D2 shRNA for 24 h, and then infected with B. pseudomallei (MOI = 10) for 12 h in the presence of DMSO or Baf A1 (10 nM). (D and E) A549 cells transiently expressing mRFP-GFP-LC3B were transfected with control or NR1D2 shRNA, and then treated with the DMSO or ATGListatin (30 μM) for 24 h and infected with B. pseudomallei (MOI = 10) for 12 h. The graph shows the quantification of mRFP + GFP + or mRFP + GFP − by taking the average number of puncta per cell. Scale bars: 5 μm. (F and G) Representative images were visualized by confocal. After transfected with control or NR1D2 shRNA, A549 cells were treated with the DMSO or ATGListatin (30 μM) for 24 h and infected with B. pseudomallei (MOI = 10) for 12 h. Quantification shows the number of B. pseudomallei in each cell. Scale bar: 5 μm. (H and I) Intracellular growth of B. pseudomallei was detected by CFU assay. A549 cells were treated as described above. Data is shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01

Article Snippet: Cells were transfected with ATG7 siRNA (sc-41447), NR1D2 shRNA (sc-61456-SH) and PNPLA2 shRNA (sc-60223-SH), which were purchased from Santa Cruz Biotechnology, using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) for 24 h. The effects of ATG7 siRNA, NR1D2 shRNA and PNPLA2 shRNA were compared with those of a nontargeting control siRNA or control shRNA.

Techniques: Infection, Transfection, Control, shRNA, Western Blot, Expressing, Colony-forming Unit Assay

Journal: Autophagy

Article Title: Burkholderia pseudomallei interferes with host lipid metabolism via NR1D2 -mediated PNPLA2/ATGL suppression to block autophagy-dependent inhibition of infection

doi: 10.1080/15548627.2020.1801270

Figure Lengend Snippet: A Schematic of the inferred mechanism by which B. pseudomallei interferes with host lipid metabolism to block autophagy-dependent inhibition of infection in human lung epithelial cells. After entry into the host cells, B. pseudomallei infection upregulates the expression of NR1D2 , which results in a decrease in PNPLA2 expression. Subsequently, PNPLA2 -dependent reduction of TG turnover and inhibition of lipophagy leads to lipid droplets accumulation, which in turn inhibits autophagic flux and promotes B. pseudomallei infection

Article Snippet: Cells were transfected with ATG7 siRNA (sc-41447), NR1D2 shRNA (sc-61456-SH) and PNPLA2 shRNA (sc-60223-SH), which were purchased from Santa Cruz Biotechnology, using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) for 24 h. The effects of ATG7 siRNA, NR1D2 shRNA and PNPLA2 shRNA were compared with those of a nontargeting control siRNA or control shRNA.

Techniques: Blocking Assay, Inhibition, Infection, Expressing

Journal: The Journal of investigative dermatology

Article Title: The Antifibrotic Effect of α2AP Neutralization in Systemic Sclerosis Dermal Fibroblasts and Mouse Models of Systemic Sclerosis.

doi: 10.1016/j.jid.2015.12.028

Figure Lengend Snippet: Figure 5. The effect of a2AP receptor in SSc dermal fibroblasts. The human dermal fibroblasts from SSc patient S4 were transfected with control or ATGL siRNA. At 48 hours after transfection, the cells were used for experiments. The expression of each protein was examined by a western blot analysis. The histogram shows quantitative representations of each protein (n ¼ 3). The data represent the mean SEM. *P < 0.01; **P < 0.05. a2AP, a2-antiplasmin; ATGL, adipose triglyceride lipase; SEM, standard error of the mean; siRNA, small interfering RNA; SSc, systemic sclerosis.

Article Snippet: SSc dermal fibroblasts were transfected with ATGL small interfering RNA (Santa Cruz Biotechnology, Dallas, TX) using Lipofectamine 2000 (Invitrogen, Camarillo, CA) according to the manufacturer’s instructions.

Techniques: Transfection, Control, Expressing, Western Blot, Small Interfering RNA