EMBO/soda-nel huggingface dataset:
Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy. (2011)
Figure 4

InlK/MVP interaction occurs in the cytosol, before actin polymerization.

A. Detection of MVP recruitment at the surface of intracellular InlK over-expressing bacteria. HeLa cells were transfected with MVP-GFP (red), infected with InlK expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy. Intra- (only green) and extracellular (cyan = green+blue) bacteria were differentially stained with anti- Listeria antibody (cf Material and Methods). Inset regions are magnified. Arrows indicate another intracellular bacterium which recruit MVP-GFP. The scale bar represents 1 µm. The right panel represents the quantification of the intracellular bacteria that recruit MVP (mean%±SEM%) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. No significant difference was found between the 4 time points. B. Detection of MVP recruitment at the surface of intracytosolic InlK over-expressing bacteria. HeLa cells were transfected with MVP-tomato (red) and YFP-CBD (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy and stained with phalloidin (blue). MVP positive bacteria were also labeled with YFP-CBD revealing that MVP was recruited by intracytosolic bacteria after the lysis of the internalization vacuole. Inset regions are magnified. The scale bar represents 1 µm. C. Kinetics of MVP and actin recruitment at the surface of InlK over-expressing bacteria. HeLa cells were transfected with MVP-tomato (red) and actin-GFP (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, and prepared for real-time video microscopy. Image series were collected every 15 min for 2 h. The left part shows an MVP positive bacterium that never recruits actin. The right part shows MVP replacement by actin around the bacterium. No colocalization of MVP-Tomato and actin-GFP was detected. Time is indicated along the Y axis. The entire image sequence can be viewed as Video S1.
Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy. (2011)
Figure 5

MVP impairs the recruitment of autophagy markers.

A. Impaired recruitment of p62 to MVP positive Listeria. HeLa cells were transfected with MVP-GFP (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue) and anti-p62 antibody (red). Inset regions are magnified. Arrows indicate independent bacteria The scale bar represents 1 µm. The vast majority of MVP-positive bacteria were completely devoid of anti-p62 labeling (95.1±2.0%; mean ± SEM from n = 3 experiments) but 4.9±2.0% (mean ± SEM from n = 3 experiments) were stained at one pole with MVP and at the other pole with p62. B. Impaired recruitment of GFP-LC3 on MVP positive Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue). Inset regions are magnified. The scale bar represents 1 µm. MVP and/or actin positive bacteria were never recognized by GFP-LC3. Arrows point to bacteria at different steps of the infection process: 1) InlK over-expressing bacterium is totally covered by MVP; 2) bacterium is partially labeled with MVP (at the poles) and actin (at the center); 3) bacterium is completely covered by actin; 4) bacterium is enclosed in an GFP-LC3 positive autophagosome. C. Kinetics of autophagy escape for MVP positive Listeria. Jeg3 cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, and prepared for real-time video microscopy. Image series were collected every 5 min for 2 h. Time is indicated along the Y axis. The left panel shows that the GFP-LC3 membranous aggregate detaches from the MVP positive Listeria. The entire image sequence can be viewed as Video S2. The right panel shows that the GFP-LC3 membranous aggregate on MVP positive bacteria does not lead to an autophagosome formation, whereas those bacteria efficiently divided. The entire image sequence can be viewed as Video S3. D. Impaired recruitment of GFP-LC3 and ubiquitin to MVP positive ΔactA Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing ΔactA (ΔactA+pADc-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with anti-ubiquitin antibody (blue) and DAPI (white). Inset regions are magnified. The scale bar represents 1 µm. E. LC3 levels in infected RAW 267.4 macrophages. Left panel: RAW 267.4 macrophages were infected with L. monocytogenes EGD (WT), ΔactA or ΔactA+InlK for 6 h. Cell total lysates were immunobloted for LC3 and actin. Western blot is representative from 3 independent experiments. Right panel: Quantification of the relative LC3-II level (mean ± SEM) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different.
Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy. (2011)
Figure 6

MVP-dependent escape from autophagy leads to increased <italic>Listeria</italic> survival.

A. InlK and ActA expression in Listeria strains used for survival assays. Total lysates of L. monocytogenes EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) grown in BHI were immunoblotted using anti-ActA and anti-InlK antibodies. B. Intracellular survival of EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in RAW 267.4 macrophages. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *. C. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP-GFP transfected Jeg3 cells. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different and are labeled here as *. D. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP-GFP transfected HeLa cells. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different and are labeled here as *. E. MVP levels in RAW 267.4 macrophages treated with MVP-siRNA. Western blot is representative from 3 independent experiments. F. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP knock-down RAW 267.4 macrophages. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.
Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease (2021)
Figure 1

Figure 1 - Increased oxidative stress in PD.

A, B. Oxidative stress biochemical markers (lipid peroxidation, ROS production) (A) and p-ERK expression (B) in tissues from 3-months old PD mice (KO) (n=3) and from wild-type (WT) mice.

C, D. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (C) and p-ERK expression (D) in PD and control (CNTR) fibroblasts (n=6).

E, F. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (E) and p-ERK expression (F) in PD and control (CNTR) myoblasts (n=4).

In all instances indicators of stress were increased in PD, compared to the respective control samples.

Data information: In each experiment at least biological triplicates were analyzed for each cell line or tissue sample; each assay was performed at least in duplicate. Data are presented as mean ± SD. Student's t-test was applied. Statistically significant comparison p-values are indicated. Fig. 1B: brightness +20%, contrast +41%.
Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease (2021)
Figure 2

Figure 2 - Modulation of autophagy impacts on stress in PD fibroblasts.

A, B. Western Blot analysis of the autophagy marker LC3 (A) and quantitative analyses (B) in untreated control fibroblasts (CNTR) and in 2 PD fibroblasts cell lines (PD1, PD2). PD cells were untreated (NT) or subjected to different treatments to modulate autophagy (starvation, STAR; rapamycin, RAPA; MK6-83; bafilomycin, BAFI). Results obtained in a third cell line (PD3) and in a control fibroblast cell line subjected to the same autophagy-modulating treatments are shown in Fig EV2.

C. ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (n=3). CNTR (white bars), CNTR after treatment (dark grey bars), PD samples (black bars) and PD after treatment (light grey bars). Physiological or pharmacological enhancement of autophagy resulted in correction of oxidative stress in PD cells, while bafilomycin treatment further increased stress in CNTR and PD cells.

Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.
Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease (2021)
Figure 3

Figure 3 - Stress inversely correlates with correction of GAA activity by rhGAA.

A, B, C. Correlations between the levels of single stress indicators and correction of GAA activity by rhGAA in 6 different PD fibroblasts. ROS production and lipid peroxidation inversely correlate with the GAA levels attained in cells after incubation with rhGAA for 4 hours.

The analysis of correlation was calculated, and the coefficient of Pearson is indicated.

D, E. Spider web charts show the trend of the four variables in each of the 6 PD cell lines analyzed after 4 (D) and 24 hours (E) of incubation with rhGAA and support the inverse relationships between the stress levels and correction of GAA activity. Multiple correlation coefficient was calculated; the increase in oxidized glutathione (GSSG level), that is complementary to the reduction of reduced glutathione (GSH), is indicated.

Data information: the scales used in D and E have been adjusted to the GAA activity at different time points.
Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease (2021)
Figure 5

Figure 5 - Effect of antioxidants on stress and ERT.

A. Effect of antioxidants on ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (3 different cell lines for each treatment, each cell line assayed at least in ducplicate). Cells were incubated for 24 hours with: idebenone (IDE) 0.5 µM; edaravone (EDA) 50 µM; N-acetylcysteine (NAC) 0.5 mM; resveratrol (RESV) 30 µM. Mean of control (CNTR) values are taken as equal to 100. The results are shown as mean ± SD.

B. Relative GAA activity increase in PD fibroblasts (3 different cell lines, each cell line assayed in duplicate) treated with rhGAA alone and with rhGAA in combination with antioxidants. The effects of rhGAA alone is taken as 100. The results are expressed as means ± SD.

C-D. Western Blot analysis of GAA isoforms and quantitative analysis of the different enzyme isoforms. The image shown is representative of three independent experiments in different PD patients.

E. Confocal immunofluorescence analysis of GAA and LAMP2; representative fibroblast cell lines (PD1). Confocal 63x images; Scale bar 50 µm; Brightness +20%. Data obtained in 2 other cell lines (PD2, PD3) are reported in Fig EV4.

F. Percent of GAA/LAMP2 colocalization in PD1. The results are expressed as means ± SD of 5 images for each condition.

G. Percent of GAA/LAMP2 colocalization; mean of the analyses performed in 3 PD fibroblast cell lines. For each PD patient, 5 images for each condition were quantified. The results are expressed as means ± SD of 5 images for each condition.

Data information: To calculate statistical significance one-way ANOVA was applied for all experiments followed by Dunnett's test. Statistically significant p-values are indicated.
Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease (2021)
Figure 6

Figure 6 - M6PR localization in PD cells and effect of antioxidants.

A, B. Fluorescence activated cell sorter (FACS) analysis of control and PD fibroblasts (n=3). A representative experiment (A) and mean values of three independent experiments (B). Data presented as mean ± SD. A Student's t-test was applied for statistical analysis.

C. FACS analysis of control and PD fibroblast show the M6PR amount at plasma membrane before and after antioxidants treatments (NAC and IDE).

D. Mean of results obtained in control (two cell lines in duplicate) and PD fibroblast (3 different cell lines, each cell line tested in duplicate in three different experiments). For each cell line, the amount of M6PR positive cells was normalized, taking that observed in not-treated fibroblasts as 1. Data presented as mean ± SD.

Data information: For statistical analysis a one-way ANOVA followed by Šídák's post hoc test was applied.
Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease (2021)
Figure 7

Figure 7 - Effect of antioxidants in the PD mouse.

A, B. Co-dosing schedule of rhGAA and NAC (A) and rhGAA and IDE (B).

C. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice = 10) and with co-dosing of rhGAA and 2 gr/Kg/day NAC (n of mice =7, dark grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues.

D. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice =4) and with co-dosing of rhGAA and 100 mg/Kg/day IDE (n of mice =6, light grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues.

E, F. Western Blot analyses of GAA and quantitative analyses of the enzyme in representative tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =2) or in combination with NAC (n of mice =3) (E) or IDE (n of mice =3) (F). In F Data are presented as mean ± SD.

G. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice = 3) or in combination with NAC (n of mice = 3).

H. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =4) or in combination with IDE (n of mice = 6).

Data information: in G and H, data are presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each tissue.

Expanded view figure legends
Multiplexed CRISPR/CAS9-Mediated Engineering of Pre-clinical Mouse Models Bearing Native Human B Cell Receptors (2020)
Figure 4

Figure 4. GC responses on Day 8 in adoptively transferred mice with rare precursor frequency.

(A) Germinal center (GC) response induced by eOD-GT8 60mer immunization in WT mice adoptively transferred with CD45.2 B cells from CLK21, CLK09, CLK19 mice on Day 8. 8-week-old CD45.1 mice were transferred with 5×105 (1 in 104) of isolated CD45.2 B cells from CLK21, CLK09 and CLK19 KI one day before the immunization with eOD-GT8 60mer, on day8, the spleenocytes were isolated and detected by FACS. First column shows the frequency of total GC (CD38loCD95+) among B cells gated from SSL, the second column shows the frequency of CD45.2 B cells among total GC, third column shows the frequency of eOD-GT8 binding B cells among GC CD45.2 B cells; fourth column shows the frequency of eOD-GT8 binding B cells that do not bind the VRC01 epitope-knockout, eOD-GT8-KO; diagrams at right show eOD-GT8 KD affinities for CLK21 (green, affinity 440 nM), CLK09 (blue, affinity 350 nM) and CLK19 (orange, affinity 1.8 µM).

(B) Quantification of GCs, CD45.2 cells and CD45.2 binder frequency in adoptively transferred mice. X-axis represents CLK21, CLK09 and CLK19 adoptively transferred mice group that immunized with eOD-GT8 60mer. Y-axis represents the frequency of total GC (left) among B cells, the frequency of CD45.2 cells within GCs and the frequency of eOD-GT8-CD45.2 binders. Each circle represents one mouse. n=5 mice/group for CLK21 and CLK09, n=3 mice/group for CLK19. Bars indicate geometric mean and geometric SD from mice in each group.

(C) Immunohistochemistry of day 8 spleen sections for host mouse receiving 5x105 isolated CD45.2 B cells from CLK09 (left) and CLK19 (right) KI mouse. Green, B220; Blue, CD3; White, CD45.2; Red, GL7.White arrows indicate the CD45.2 B cells.
Multiplexed CRISPR/CAS9-Mediated Engineering of Pre-clinical Mouse Models Bearing Native Human B Cell Receptors (2020)
Figure 6

Figure 6. Duration of GC response in CD45.2 adoptively transferred mice

(A) CD45.2 frequency among GCs induced by eOD-GT8 60 mer at different time points. Adoptively transferred mice with 1 in 104 of precursor frequency were immunized with eOD-GT8 60mer. On days 8, 15 and 36, the splenic B cells were isolated to measure the CD45.2 cell frequency in GC B cells by FACS using the markers as in Figure 5B mentioned above by FACS. Gated plots represent the CD45.2 frequency among GC B cells.

(B) Quantification of CD45.2 frequency among GCs at three time points. X-axis represents adoptively transferred mice groups with different precursor frequency (1 in 104, 2 in 105, 3 in 106) at three time point, y axis represents CD45.2 frequency among GC. Each circle represents one mouse. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. Bars indicate mean ± SD from mice in each group.

(C) IgG titers detected by ELISA. eOD-GT8 or eOD-GT8 KO soluble proteins were used to coat ELISA plates. Sera from adoptively transferred mice at different time points were used to detect by ELISA. Bars indicate geometric mean and geometric SD from mice in each group. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. X-axis represents precursor frequency, and Y-axis represents the change of area under curve (AUC coated eOD-GT8 -AUC coated eOD-GT8 KO). Each circle represents one mouse.
A solid-phase transfection platform for arrayed CRISPR screens (2019)
Figure 2

Figure 2: Solid phase transfection for delivery of synthetic guide RNAs

A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 24, 48 and 72 hours and imaged after DNA staining with Hoechst. Green arrowheads indicate examples of prometaphase arrested cells, and the red arrowheads indicate examples of dead cells due to Plk1 downregulation. Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments. Scale bar, 20 μm.

B) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst.

C) Quantification of nuclear size measurements from Fig 2B. Data derived from 3 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.

D) Solid phase transfection of nontargeting (scrambled), GOLGA2 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Golga2 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm.

E) Quantification of experiments in Fig 2D. Data derived from 3 independent experiments. P value (scrambled vs GOLGA2) < 2e-16, Mann-Whitney U test.

F) Solid phase transfection of nontargeting (scrambled), MKI67 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm.

G) Quantification of experiments in Fig 2F. Data derived from 3 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test.

H) Comparison of phenotypic penetrance with liquid and solid phase transfection. RPE-1, HEK293T, NCI-H358 and NCI-N87 cells were transfected with scrambled or POLR2A targeting gRNAs in in two different cell numbers (2250 or 20000 cells/well). 5 days post transfection cell viability were measured by CellTiter-Glo. Boxplots represent values from at least 3 independent experiments containing 3 technical replicates.

I) Cell viability measurements after solid phase transfection targeting POLR2A in a panel of cell lines. Cas9 expressing cell lines were transfected with scrambled and POLR2A targeting gRNA and cell viability was assessed after 5 days. The raw values are background subtracted and normalized to the mock controls. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.
A solid-phase transfection platform for arrayed CRISPR screens (2019)
Figure 3

Figure 3: Solid phase transfection for delivery of Cas9/guide RNA ribonucleoprotein complexes

A) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. Scale bar, 50μm.

B) Quantification of experiments in Fig 3A. Data derived from 2 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.

C) Solid phase transfection of nontargeting (scrambled), MKI67 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm.

D) Quantification of experiments in Fig 3C. Data derived from 2 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test.

E) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.
A solid-phase transfection platform for arrayed CRISPR screens (2019)
Figure 4

Figure 4: Arrayed CRISPR screens using solid phase transfection

A) Viability of NCI-N87 and NCI-H358 cells upon transfection of 45 different gRNAs by solid phase transfection. Cas9 expressing NCI-N87 and NCI-H358 cell were seeded on pre-coated plates. 5 days post transfection, cell viability in each well were measured by CellTiter-Glo. Values were background subtracted, normalized to scrambled controls and plotted against each other. Blue dots represent scrambled controls, whereas the yellow dots represent POLR2A gRNAs. Green dots represent the genes that affect the viability in a cell line dependent manner. Results are representative of 3 independent experiments.

B, C) Logarithm of the odds (LOD) scores of cancer associated gene KOs in NCI-N87 and NCI-H358 cells derived from screens with gRNAs (B) and RNPs (C). Bottom panels show distribution of positive (POLR2A) and negative (scrambled) controls for each experiment. LOD score of 3 is highlighted with a dashed line, indicating statistical significance.