Journal: eLife

Article Title: A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells

doi: 10.7554/eLife.08474

Figure Lengend Snippet: HAP1 cells were co-transfected with plasmids encoding Cas9, a target gene-specific sgRNA, and GFP. Successfully transfected (GFP + ) cells were sorted 24–72 hr post-transfection and re-plated. 5–7 days post-transfection, cells were stained for ULBP1 expression and analyzed by flow cytometry. Examples are shown for ULBP1 ( A ), RBM4 and ATF4 ( B ), and SPCS1 ( C ). DOI: http://dx.doi.org/10.7554/eLife.08474.007

Article Snippet: Mutant HAP1 cells were generated by transiently co-transfecting cells with a Cas9 expression vector (pMJ918, a gift from Jennifer Doudna), an sgRNA expression vector (Addgene plasmid #41824, a gift from George Church), and a GFP expression vector using Lipofectamine 2000.

Techniques: Transfection, Staining, Expressing, Flow Cytometry

Journal: EMBO Reports

Article Title: Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling

doi: 10.15252/embr.201947734

Figure Lengend Snippet: Western blot analyses of glial cells infected with shRNA against Nf‐1 and Tp53 , and then with Cas9 and sgRNAs against Atg7 .

Article Snippet: For CRISPR/Cas9‐mediated gene editing in MEFs, the sgRNA vectors described above (Addgene #41824) were transfected along a Cas9 vector (hCas9, gift from George Church, Addgene #41815) using Lipofectamine 2000.

Techniques: Western Blot, Infection, shRNA

Journal: EMBO Reports

Article Title: Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling

doi: 10.15252/embr.201947734

Figure Lengend Snippet: The following experiments were performed in glial sh Nf‐1 /sh Tp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sg Atg7 #1 or #2). Spinning disc confocal live cell imaging of Alexa 555‐EGF (555‐EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/ml 555‐EGF for the indicated durations. Scale bar: 10 μm. Immunofluorescence staining of EGFR following 5‐ or 15‐min stimulation with 20 ng/ml EGF. Scale bar: 20 μm. Quantification of EGFR vesicles in a perinuclear region (within 30 μm diameter of the centre of the nucleus) (in B). Cells were stimulated with 20 ng/ml 555‐EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm. Quantification of percentage of total EGFR vesicles that colocalise with 555‐EGF (in D). Cells stably expressing mCherry‐Rab5 were stimulated with 20 ng/ml EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm. Pearson's colocalisation coefficient between mCherry‐Rab5 and EGFR (in F). Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two‐tailed Student's t ‐test: NS P > 0.05, * P < 0.05, ** P < 0.01.

Article Snippet: For CRISPR/Cas9‐mediated gene editing in MEFs, the sgRNA vectors described above (Addgene #41824) were transfected along a Cas9 vector (hCas9, gift from George Church, Addgene #41815) using Lipofectamine 2000.

Techniques: Expressing, Control, Live Cell Imaging, Immunofluorescence, Staining, Stable Transfection, Two Tailed Test

Journal: EMBO Reports

Article Title: Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling

doi: 10.15252/embr.201947734

Figure Lengend Snippet: The following experiments were performed in glial sh Nf‐1 /sh Tp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sg Atg7 #1 or #2). Cells transiently expressing YFP‐Galectins (YFP‐Gal3, YFP‐Gal8, or YFP‐Gal9) were stimulated with 2 ng/ml EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with YFP‐Galectins (in A). Cells transiently expressing YFP‐Gal8 were stimulated with 20 ng/ml Alexa 555‐EGF (555‐EGF) for 15 min. Scale bar: 10 μm. Quantification of the percentage of YFP‐Gal8‐labelled vesicles that colocalise with 555‐EGF in control or ATG7‐deficient cells (in C). Cells transiently expressing YFP‐Gal8 were treated 100 μM monensin for 1 h and stimulated with 2 ng/ml EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with YFP‐Gal8 upon monensin treatment (in E). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two‐tailed Student's t ‐test: NS P > 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For CRISPR/Cas9‐mediated gene editing in MEFs, the sgRNA vectors described above (Addgene #41824) were transfected along a Cas9 vector (hCas9, gift from George Church, Addgene #41815) using Lipofectamine 2000.

Techniques: Expressing, Control, Immunofluorescence, Staining, Two Tailed Test

Journal: EMBO Reports

Article Title: Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling

doi: 10.15252/embr.201947734

Figure Lengend Snippet: The following experiments were performed in glial sh Nf‐1 /sh Tp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sg Atg7 #1 or #2). Cells stably expressing GFP‐LC3 were treated with 100 μM monensin in the presence or absence of bafilomycin A1 (20 nM) for 1 h and stimulated with 2 ng/ml EGF for 15 min before fixation and immunofluorescence staining against EEA1. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with GFP‐LC3 (in A). Cells were treated for 1 h with 100 μM monensin and then stimulated with 2 ng/ml EGF for 15 min. LAMP2 and EEA1 were then detected by immunofluorescence staining. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of the percentage of total EEA1 vesicles that colocalise with LAMP2 (in C). Cells were treated with 2 ng/ml EGF for 15 min before immunofluorescence against endogenous ATG16L1 and EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 puncta that colocalise with ATG16L1 (in E). Cells stably expressing Flag‐S‐ATG16L1 were treated for 1 h with either 100 μM monensin or 30 μM Dynasore, and then stimulated with 2 ng/ml EGF for 15 min. Cells were then stained by immunofluorescence against EEA1 and Flag tag. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with Flag‐S‐ATG16L1 (in G). Untreated control or sg Atg7 cells, or control cells pre‐treated with 100 μM monensin 1 h, stably expressing Flag‐S‐ATG16L1 were stimulated 20 ng/ml Alexa 555‐EGF (555‐EGF) for 15 min before fixation and immunofluorescence staining against Flag tag and EEA1. Cells were then imaged by structured illumination microscopy (SIM), and images were reconstructed in Nikon Elements software. Scale bar: 10 μm. Quantification of the percentage of EGF‐EEA1 colocalised vesicles that stained triple‐positive with ATG16L1 by SIM (in I). Due to the low‐throughput nature of this assay, the following cell numbers were counted: control untreated (9 cells), sg Atg7 #1 (10 cells), sg Atg7 #2 (9 cells) and control + monensin (11 cells). Cells were treated with 2 ng/ml EGF for 15 min before immunofluorescence against endogenous WIPI2 and EEA1. Scale bar: 10 μm. Quantification of percentage of total EEA1 puncta that colocalise with WIPI2 (in K). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two‐tailed Student's t ‐test: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For CRISPR/Cas9‐mediated gene editing in MEFs, the sgRNA vectors described above (Addgene #41824) were transfected along a Cas9 vector (hCas9, gift from George Church, Addgene #41815) using Lipofectamine 2000.

Techniques: Expressing, Control, Stable Transfection, Immunofluorescence, Staining, FLAG-tag, Microscopy, Software, Two Tailed Test

Journal: EMBO Reports

Article Title: Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling

doi: 10.15252/embr.201947734

Figure Lengend Snippet: The following experiments were performed in glial sh Nf‐1 /sh Tp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sg Atg7 #1 or #2). Cells were treated for 15 min with 2 ng/ml EGF before fixation and immunofluorescence staining against EEA1 and p‐TBK1. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with p‐TBK1 (in A). Cells stably expressing Flag‐S‐ATG16L1 were pre‐treated for 1 h with TBK1 inhibitors (100 μM MRT68601 or 5 μM momelotinib). Control cells were also treated 100 μM monensin for 1 h as indicated. All cells were stimulated for 15 min with 2 ng/ml EGF followed by fixation and immunofluorescence staining against Flag tag and EEA1. Scale bar: 10 μm. Quantification of total EEA1 vesicles that colocalise with Flag‐S‐ATG16L1 (in C). Western blotting of sh Nf‐1 /sh Tp53 glial cells expressing gRNA sequences targeting Gal8 . Control and sg Gal8 cells stably expressing Flag‐S‐ATG16L1 were treated 100 μM monensin for 1 h and stimulated with 2 ng/ml EGF for 15 min before fixation and immunofluorescence staining against EEA1 and Flag tag. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of percentage of total EEA1 vesicles that colocalise with Flag‐S‐ATG16L1 in sg Gal8 cells (in F). Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two‐tailed Student's t ‐test: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For CRISPR/Cas9‐mediated gene editing in MEFs, the sgRNA vectors described above (Addgene #41824) were transfected along a Cas9 vector (hCas9, gift from George Church, Addgene #41815) using Lipofectamine 2000.

Techniques: Expressing, Control, Immunofluorescence, Staining, Stable Transfection, FLAG-tag, Western Blot, Two Tailed Test

Journal: EMBO Reports

Article Title: Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling

doi: 10.15252/embr.201947734

Figure Lengend Snippet: The following experiments were performed in sh Nf‐1 /sh Tp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sg Atg7 #1 or #2). Cells were treated with 20 ng/ml Alexa 555‐EGF (555‐EGF) for the indicated times before fixation. 555‐EGF was detected using the ImageXpress high‐content imaging platform, and representative images of 555‐EGF are shown. Scale bar: 100 μm. Following high‐content imaging of 555‐EGF and DAPI, 555‐EGF uptake relative to cell number was quantified and made relative in each cell line to background readings from unstimulated cells ( t = 0 min) (in A). Control or sg Atg7 glial sh Nf‐1 /sh Tp53 cells were either untreated, serum starved (‐FBS) for 4 h, or amino acid starved (‐AA) for 2 h and LysoSensor probe added for 1 h. Shown are quantifications of LysoSensor fluorescence intensity per cell normalised to untreated control cells. Cells were stimulated with 20 ng/ml EGF for the indicated times before EGFR levels were analysed by Western blotting. Densitometry analyses of Western blotting showing EGFR degradation with measurements made relative to EGFR levels in unstimulated cells ( t = 0 min) (in D). Cells were stimulated with 20 ng/ml 555‐EGF for 15 min before immunofluorescence staining against endogenous Rab7. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of Pearson's colocalisation coefficient between 555‐EGF and Rab7 (in F). Schematic representation of cell surface protein biotinylation assay to assess endocytosis and recycling rates (see Fig C and D). Cells stably expressing mCherry‐Rab4 were stimulated with 20 ng/ml EGF for 5 or 15 min and then fixed and stained by immunofluorescence against EGFR. White arrows indicate colocalisation. Scale bar: 10 μm. Quantification of Pearson's colocalisation coefficient between mCherry‐Rab4 and EGFR (in I). Cells were stimulated with Alexa 555‐transferrin (555‐Tfn, 20 ng/ml) for 15 min and chased with media without transferrin for 15 min. Cells were then stained by immunofluorescence against Rab11. Scale bar: 10 μm. Quantification of Pearson's colocalisation coefficient between 555‐Tfn and Rab11 (in K). Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two‐tailed Student's t ‐test: NS P > 0.05, * P < 0.05, ** P < 0.01.

Article Snippet: For CRISPR/Cas9‐mediated gene editing in MEFs, the sgRNA vectors described above (Addgene #41824) were transfected along a Cas9 vector (hCas9, gift from George Church, Addgene #41815) using Lipofectamine 2000.

Techniques: Expressing, Control, Imaging, Fluorescence, Western Blot, Immunofluorescence, Staining, Cell Surface Biotinylation Assay, Stable Transfection, Two Tailed Test