Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: GORAB promotes embryonic lung maturation through antagonizing AKT phosphorylation, versican expression and mesenchymal cell migration

doi: 10.1096/fj.201902075R

Figure Lengend Snippet: (A) Appearance of alveolar-like structure formed by co-culturing A549 epithelial cells with primary fibroblasts isolated from lungs of E18.5 Gorab+/+ or Gorab−/− fetuses for indicated durations. (B) Quantifications of the thickness (left) and height (middle) of cell ridge and the area of pockets (right) at 72 hours of co-culturing. (C) Representative distribution of cells in 72-hour co-cultures by immunofluorescence labeling of A549 (CellTracker dye CMTPX, red) and fibroblasts (vimentin, VIM, green). (D - E) Appearance (D) and quantification (E) of alveolar-like structure formed by co-culturing A549 epithelial cells with primary fibroblasts isolated from the lungs of wild type E18.5 Gorab+/+ fetuses and transfected with Vcan-V2 cDNA or Vector. Mock indicates non-transfected cells. Cells were examined 48 hours after transfection. (F - G) Appearance (F) and quantification (G) of alveolar-like structure formed by co-culturing A549 epithelial cells with primary fibroblasts isolated from the lungs of E18.5 Gorab−/− fetuses and transfected with Vcan siRNA or non-targeting (NT) siRNA. Mock indicates non-transfected cells. Cells were examined 48 hours after transfection. Data are shown as means ± SD (n ≥ 3 independent experiments). Student’s t-test and Bonferroni’s multiple comparison test, one-way ANOVA. ∗ P < 0.05 vs. corresponding controls. Scale bars, 500 μm (A), 100 μm (C), 250 μm (D, F).

Article Snippet: Mouse Vcan cDNA (V2) was obtained from Origene (MR225930, Rockville, MD) and transfected (1 μg per well of 12-well plate) to mouse primary lung fibroblasts as described for siRNA.

Techniques: Isolation, Immunofluorescence, Labeling, Transfection, Plasmid Preparation

Journal: Developmental cell

Article Title: LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.

doi: 10.1016/j.devcel.2023.05.009

Figure Lengend Snippet: Figure 2. LC3B is not recruited to LDs by known factors (A) Confocal imaging of EGFP-LC3B, mRFP-P62, and LDs in differentiated 3T3-L1 adipocytes. Cells were virally transfected with EGFP-LC3B and mRFP-P62 and incubated in EBSS for 48 h. Scale bars,10 mm (5 mm in insets). (B) Schematic representation illustrating the recruitment of EGFP-LC3B alone or with mRFP-P62 on LDs. The percentage of LDs of each phenotype is written below the corresponding schematic representation. Quantifications are the average from three independent experiments. (C) Top: western blot of differentiated 3T3-L1 adipocytes virally co-transfected with EGFP-LC3B and with either ATG5 shRNA or non-targeting shRNA for 24 h, and then incubated in EBSS for 24 h. Bottom: quantification of ATG5 expression from three independent experiments Student’s unpaired t test is used (**p < 0.001). (D) Confocal imaging of LDs in differentiated 3T3-L1 adipocytes treated as described in (C). Scale bars, 10 mm (5 mm in insets). (E) Top: percentage of cells with EGFP-LC3B-positive LDs. Bottom, percentage of EGFP-LC3B-positive LDs per cell. Quantifications are from three independent experiments. Student’s unpaired t test is used (***p < 0,0001, ns p > 0,05). (F) Confocal imaging of EGFP-LC3B and LDs in differentiated 3T3-L1 adipocytes virally transfected with EGFP-LC3B and incubated in EBSS alone or EBSS containing Spautin-1 for 48 h. Scale bars,10 mm (5 mm in insets). (G) Left, percentage of cells with EGFP-LC3B-positive LDs. Right, percentage of EGFP-LC3B-positive LDs in the cell. Quantifications are from three independent experiments done as described in (F) Student’s unpaired t test is used (**p < 0.001, ns p > 0.05). (H) Western blot of lysate and LD fractions of differentiated adipocytes incubated in EBSS alone or EBSS containing Spautin-1 for 48 h. (I) Confocal imaging of EGFP-LC3B and LDs in differentiated 3T3-L1 adipocytes virally transfected with EGFP-LC3B and incubated in EBSS alone or EBSS containing bafilomycin A for 48 h. Scale bars,10 mm (5 mm in insets). To right, up: percentage of EGFP-LC3B-positive LDs in the cell. Down: percentage of cells with EGFP-LC3B-positive LDs. Quantifications are from three independent experiments. Student’s unpaired t test is used (* p < 0,05, ns p > 0,05). (J) Schematic representation of the impact of swelling of intracellular organelles by incubating the cells with a hypotonic media. (K) Time-lapse imaging experiment performed on Huh7 cells that were transfected with EGFP-LC3B, treated with oleic acid for 24 h, and then incubated in EBSS for 48 h. At time 0, a hypotonic media was added to induce cell swelling. Imaging was done at the indicated times. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PcDNA ATGL Carole Sztalryd, University of Maryland School of Medicine N/A LentiCRISPRv2-ATG5 Edward Campbell Addgene # 99573 LentiCRISPRv2-Beclin1 Edward Campbell Addgene # 99574 LAMP1-mRFP-FLAG David Sabatini Addgene # 34611 pLAMP1-mCherry Amy Palmer Addgene # 45147 pDEST-CMV 3xFLAG-LC3A-GFP Robin Ketteler Addgene # 123106 pDEST-CMV 3xFLAG-LC3C-GFP Robin Ketteler Addgene # 123110 psPAX2 Didier Trono Addgene # 12260 pBABE-puro mCherry-EGFP-LC3B Jayanta Debnath Addgene # 22418 shRNA Atg3 (h) Plasmide Santa Cruz biotech Cat# # Sc-72582-SH Software and algorithms Fiji https://fiji.sc/ N/A Adope Illustrator 2023 https://www.adobe.com N/A GraphPad Prism 7.0a https://www.graphpad.com N/A MATLAB https://fr.mathworks.com N/A

Techniques: Imaging, Transfection, Incubation, Western Blot, shRNA, Expressing

Journal: Developmental cell

Article Title: LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.

doi: 10.1016/j.devcel.2023.05.009

Figure Lengend Snippet: Figure 3. ATG3 is recruited to lipid droplets during long-term nutrient starvation (A) Immunofluorescence staining of LC3B, ATG3, and LDs in differentiated 3T3-L1 adipocytes incubated in DMEM or EBSS for 48 h. (B) Immunofluorescence staining of ATG3 and LDs in differentiated 3T3-L1 adipocytes incubated in EBSS containing 3MA for 48 h. (C) Percentage of ATG3-positive LDs per cell. Student’s unpaired t test is used (ns p > 0,05). (D) Confocal imaging of Huh7 cells co-transfected with EGFP-LC3B and ATG3-dsRed. Cells were treated with OA to induce LDs and then incubated in EBSS for 48 h. Scale bars, 10 mm (5 mm in insets). (E) Confocal imaging of LDs in differentiated 3T3-L1 adipocytes virally co-transfected with EGFP-LC3B and an ATG3 shRNA or EGFP-LC3B and the non-targeting shRNA. Cells were incubated in EBSS for 48 h after transfection. Scale bars, 10 mm (5 mm in insets). (F) Western blot of cells treated as described in (E). (G) The bar graph shows the quantification of ATG3 expression from three western blots of cells treated as described in (E). Student’s unpaired t test is used (*** p < 0,0001). (H) Right, percentage of cells with EGFP-LC3B-positive LDs. Left, percentage of EGFP-LC3B-positive LDs per cell. Quantifications are from three independent experiments. Student’s unpaired t test is used (*** p < 0,0001). (I) Immunofluorescence staining of LC3B and PLIN1 in differentiated adipocytes stably transfected with mATG3 WT, mATG3 K11W, or mATG3 V15K. Cells were incubated in EBSS for 48 h then fixed and stained (LC3B in green, PLIN1 in magenta). Scale bars, 10 mm (5 mm in insets). (J) Right, percentage of cells with LC3B-positive LDs. Left, percentage of LC3B-positive LDs per cell. Quantifications are from three independent experiments. An ordinary one-way ANOVA test was used (*** p < 0,0001). See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PcDNA ATGL Carole Sztalryd, University of Maryland School of Medicine N/A LentiCRISPRv2-ATG5 Edward Campbell Addgene # 99573 LentiCRISPRv2-Beclin1 Edward Campbell Addgene # 99574 LAMP1-mRFP-FLAG David Sabatini Addgene # 34611 pLAMP1-mCherry Amy Palmer Addgene # 45147 pDEST-CMV 3xFLAG-LC3A-GFP Robin Ketteler Addgene # 123106 pDEST-CMV 3xFLAG-LC3C-GFP Robin Ketteler Addgene # 123110 psPAX2 Didier Trono Addgene # 12260 pBABE-puro mCherry-EGFP-LC3B Jayanta Debnath Addgene # 22418 shRNA Atg3 (h) Plasmide Santa Cruz biotech Cat# # Sc-72582-SH Software and algorithms Fiji https://fiji.sc/ N/A Adope Illustrator 2023 https://www.adobe.com N/A GraphPad Prism 7.0a https://www.graphpad.com N/A MATLAB https://fr.mathworks.com N/A

Techniques: Staining, Incubation, Imaging, Transfection, shRNA, Western Blot, Expressing, Stable Transfection

Journal: Developmental cell

Article Title: LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.

doi: 10.1016/j.devcel.2023.05.009

Figure Lengend Snippet: Figure 4. ATG3 better binds to model LDs enriched in PE (A) Confocal imaging of triolein droplets before and after ATG3-YFP addition. Scale bars, 100 mm. (B) Top: schematic illustration of triolein-in-buffer droplets decorated by different phospholipid densities ([1/1] PC/PE), reported by rhodamine-PE (Rho-PE). Bottom: confocal imaging of triolein-in-buffer droplets with different phospholipid coverage, ranging from 0.005% to 0.2% (w/w to triolein) before and after ATG3 addition. Scale bars, 100 mm. Line profiles show the intensity levels of ATG3-YFP and Rho-PE on droplets depicted in the inset. (C) ATG3-YFP recruitment to triolein droplets as a function of the phospholipid density, reported by Rho-PE. The concentration at half of maximum binging is depicted in the main figure. Concentration at half of maximum binding C1/2 is shown in red. The inset figure shows the different recruitment profiles of Atg3-YPF depending on the PC/PE ratio. (D) The characteristic concentration C1/2 of ATG3-YFP binding from experiments done as described in (B) for the indicated PC/PE ratio. (E) Western blot of untagged ATG3 recombinant protein bound to liposomes and artificial LDs in the top fraction of flotation assays. (F) Schematic representation of the droplet-embedded vesicle (DEV) system. (G) Confocal imaging of a DEV made of 7/3 PC/PE and incubated with ATG3-YFP. Scale bars, 10 mm. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PcDNA ATGL Carole Sztalryd, University of Maryland School of Medicine N/A LentiCRISPRv2-ATG5 Edward Campbell Addgene # 99573 LentiCRISPRv2-Beclin1 Edward Campbell Addgene # 99574 LAMP1-mRFP-FLAG David Sabatini Addgene # 34611 pLAMP1-mCherry Amy Palmer Addgene # 45147 pDEST-CMV 3xFLAG-LC3A-GFP Robin Ketteler Addgene # 123106 pDEST-CMV 3xFLAG-LC3C-GFP Robin Ketteler Addgene # 123110 psPAX2 Didier Trono Addgene # 12260 pBABE-puro mCherry-EGFP-LC3B Jayanta Debnath Addgene # 22418 shRNA Atg3 (h) Plasmide Santa Cruz biotech Cat# # Sc-72582-SH Software and algorithms Fiji https://fiji.sc/ N/A Adope Illustrator 2023 https://www.adobe.com N/A GraphPad Prism 7.0a https://www.graphpad.com N/A MATLAB https://fr.mathworks.com N/A

Techniques: Imaging, Concentration Assay, Binding Assay, Western Blot, Recombinant, Liposomes, Incubation

Journal: Developmental cell

Article Title: LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.

doi: 10.1016/j.devcel.2023.05.009

Figure Lengend Snippet: Figure 5. ATG3 lipidates LC3 to purified and artificial LDs (A) Confocal imaging of purified adipocyte LDs in HKM buffer containing Alexa 488-LC3B, in the presence or absence of the lipidation reaction components ATG7, ATG3, and ATP. (B) LDs from the previous experiment are collected and analyzed using SDS-PAGE in a stained Coomassie blue. (C) Confocal imaging of triolein-in-buffer droplets decorated by PC/PE (7/3) incubated with Alexa 488-LC3B, then ATG7 and ATP. No lipidation occurred. When ATG3 was subsequently added, lipidation occurred on the artificial LDs (arrows show examples). (D) Artificial LDs from the previous experiment are collected and analyzed using SDS-PAGE in a stained Coomassie blue. (E) Triolein-in-buffer droplets decorated with PC/PE at different monolayer phospholipid densities (based on Rho-PE signal) are imaged using confocal mi- croscopy after being incubated with Alexa 647-LC3B and Atg3/Atg3-YFP (80/20), ATP, and ATG7. (F) Confocal imaging of triolein-in-buffer droplets decorated by PC/PE (7/3) at different monolayer phospholipid densities varied from 0.005% to 0.2% (w/w to triolein). They are incubated with Alexa 488-LC3B and Atg3 (80/20), ATP, and ATG7. (G) Quantification of F. LC3B-Alexa 488 lipidation to triolein droplets as a function of the phospholipid density. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PcDNA ATGL Carole Sztalryd, University of Maryland School of Medicine N/A LentiCRISPRv2-ATG5 Edward Campbell Addgene # 99573 LentiCRISPRv2-Beclin1 Edward Campbell Addgene # 99574 LAMP1-mRFP-FLAG David Sabatini Addgene # 34611 pLAMP1-mCherry Amy Palmer Addgene # 45147 pDEST-CMV 3xFLAG-LC3A-GFP Robin Ketteler Addgene # 123106 pDEST-CMV 3xFLAG-LC3C-GFP Robin Ketteler Addgene # 123110 psPAX2 Didier Trono Addgene # 12260 pBABE-puro mCherry-EGFP-LC3B Jayanta Debnath Addgene # 22418 shRNA Atg3 (h) Plasmide Santa Cruz biotech Cat# # Sc-72582-SH Software and algorithms Fiji https://fiji.sc/ N/A Adope Illustrator 2023 https://www.adobe.com N/A GraphPad Prism 7.0a https://www.graphpad.com N/A MATLAB https://fr.mathworks.com N/A

Techniques: Imaging, SDS Page, Staining, Incubation

Journal: International Journal of Nanomedicine

Article Title: Ginseng-berry-mediated gold and silver nanoparticle synthesis and evaluation of their in vitro antioxidant, antimicrobial, and cytotoxicity effects on human dermal fibroblast and murine melanoma skin cell lines

doi: 10.2147/IJN.S118373

Figure Lengend Snippet: Comparative study of the effect of GBAuNPs, GBE and HAuCl 4 ·3H 2 O on cell viability and on the morphology of HDF and B16 cells. Notes: Optical microscopy images ( A ) of HDF and B16 cell lines (40× magnification) at 72 h after treated with 1–100 μg/mL of GBAuNPs. Treated HDF and B16 cells with AuNPs (10 and 100 μg/mL) could be identified by dark dense clusters which are indicated with arrows; no cluster was found in control groups. Cytotoxicity effect of GBE and GBAuNPs on HDF ( B ) and B16BL6 ( C ) cell lines; GBAuNPs did not exhibit a significant cytotoxic effect on HDF and B16 cells. Cell viability was determined by MTT assay. Data are expressed as a percentage of sample-treated control and presented as mean ± SEM of three separate experiments. B16, murine melanoma B16BL6 cells. Abbreviations: AuNPs, gold nanoparticles; GBAuNPs, AuNPs from ginseng berry; GBE, ginseng berry extract; HDF, human dermal fibroblast; SEM, standard error of the mean.

Article Snippet: HDF and B16 cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea).

Techniques: Microscopy, Control, MTT Assay

Journal: International Journal of Nanomedicine

Article Title: Ginseng-berry-mediated gold and silver nanoparticle synthesis and evaluation of their in vitro antioxidant, antimicrobial, and cytotoxicity effects on human dermal fibroblast and murine melanoma skin cell lines

doi: 10.2147/IJN.S118373

Figure Lengend Snippet: Comparative study of the effect of GBAgNPs, GBE and AgNO 3 on cell viability and on the morphology of HDF and B16 cells. Notes: Optical microscopy images ( A ) of HDF and B16 cell lines (40× magnification) at 72 h after treated with 1–100 μg/mL of GBAgNPs. Treated HDF and B16 cells with silver (10 μg/mL) could be identified by dark dense clusters which are indicated with arrows; no cluster was found in control groups. Cytotoxicity effect of GBE, GBAgNPs, and silver salts on HDF ( B ) and B16 ( C ) cell lines; cell viability decreased with an increase in the concentration of GBAgNPs. Cell viability was determined by MTT assay. Data are expressed as a percentage of sample-treated control and presented as mean ± SEM of three separate experiments. Abbreviations: B16, murine melanoma B16Bl6 cells; GBAgNPs, silver nanoparticles from ginseng berry; GBE, ginseng berry extract; HDF, human dermal fibroblast; MTT, 3-(4,5-dimethyl-thiazol-2yl)-2, 5-diphenyl tetrazolium bromide; SEM, standard error of the mean.

Article Snippet: HDF and B16 cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea).

Techniques: Microscopy, Control, Concentration Assay, MTT Assay

Journal: RSC Advances

Article Title: In vitro and in vivo biocompatibility and inflammation response of methacrylated and maleated hyaluronic acid for wound healing

doi: 10.1039/d0ra06025a

Figure Lengend Snippet: Cell proliferation investigation of HUVEC (a), HDF (b) and HAD-MSC (c) seeded on MEHA and MAHA at long time (1, 3 and 7 days). Cell proliferation was performed by CCK8 assay using manufacturer's protocol. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, no significant difference.

Article Snippet: HDF, HAD-MSC and HUVEC (ScienCell – California, USA) were cultured in 75 cm 2 cell culture flask.

Techniques: CCK-8 Assay