EMBO/soda-nel huggingface dataset:
A solid-phase transfection platform for arrayed CRISPR screens (2019)
Figure 5

Figure 5: Solid phase transfection in primary cells derived from human tumors

A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting RNP complexes into human primary lung fibroblasts (HLF-1), primary lung adenocarcinoma cells (LAD-1) or RPE-1TP53 -/- cells. 72 hours post transfection cells were stained with Hoechst and imaged. Boxplots represent values from 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs Plk1) < 0.05. In the right panels, representative images for each cell line is shown after RNP transfection targeting PLK1. Arrowheads indicate the cells that are arrested in prometaphase due to Plk1 downregulation. Scale bars indicate 20μm.

B) Two human primary lung fibroblasts (HLF) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma (LSC) or lung adenocarcinoma (LAD) 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.

Expanded View Figure legends:
Tollip coordinates Parkin-dependent trafficking of mitochondria-derived vesicles (2020)
Figure 1

Figure 1: Tollip mediates the trafficking of TOM20+ve MDVs.

(A) siRNA knockdown of Tollip in SH-SY5Y cells was performed over 72 hours, with cells reverse transfected with siRNA oligos specific for Tollip (or mock) for 24 hours before a second transfection was performed for a further 48 hours. Knockdown was confirmed by western blotting.

(B) After knockdown of Tollip, SH-SY5Y cells were treated with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM Antimycin A (AA) alone for 2 hours, then fixed and stained for PDH E2/E3bp (green), TOM20 (red) and nuclei (blue). Images were captured using widefield immunofluorescence microscopy. Zoom insets represent 4x magnification and arrowheads indicate TOM20-positive MDVs.

(C, D) The number of TOM20+ve/PDH-ve MDVs (C) or PDH+ve/TOM20-ve MDVs (D) per cell were quantified from immunofluorescence images captured (n = 30 cells per condition from 2 independent experiments). Statistical significance was determined using a Two-way ANOVA (Tukey). Centre line indicates the mean and error bars represent SEM.

(E) SH-SY5Y cells were transfected with GFP-labelled Tollip for 24 hours then treated with AO, AO/100 μM bafilomycin A1 (AO/BfnA1) or AA alone for 2 hours prior to fixation. Cells were then immunostained for GFP (green), PDH E2/E3bp (red) and TOM20 (blue) and imaged by widefield immunofluorescence microscopy. GFP-Tollip was observed on TOM20+ve/PDH-ve MDVs (arrowheads). Zoom insets represent 7x magnification.

(F) Images captured using super-resolution radial fluctuations (SRRF) microscopy showing both TOM20 (arrowheads) and PDH (arrows) MDVs in SH-SY5Y cells after Tollip siRNA knockdown. Left side images are the first in each of the original widefield image stacks. Scale bar for zoom images is 0.5 μm.

Data information: Scale bars are 10 μm, unless otherwise stated.
Tollip coordinates Parkin-dependent trafficking of mitochondria-derived vesicles (2020)
Figure 2

Figure 2: Parkin and Tollip interact on a vesicular compartment.

(A) TOM20-positive MDV formation was induced in SH-SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20+ve/Cytochrome c (Cyt c)-ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads).

(B) SH-SY5Y cells were transfected with GFP-Tollip for 24 hours, then treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 hours prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used.

(C) GFP-Tollip/Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3-6 GFP-transfected cells/condition, per experiment (n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin.

(D) SH-SY5Y cells expressing GFP-Tollip were immunostained for GFP (green), Parkin (red), and Rab7a (blue). Arrowheads indicate areas of colocalisation between all three markers.

(E-F) SH-SY5Y cells were transfected with GFP-Tollip R78A or CUE mutant for 24 hours, then treated with AO or AO/BfnA1 for 2 hours prior to fixation and immunostaining, as described above for (B).

(G) The percentage of GFP-Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/ condition, per experiment (n = 3; *p=0.0162, **p=0.0034, ****p<0.0001).

(H) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0382, **p=0.0068).

(I) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0232, **p=0.0062).

(J) The percentage of GFP-Tollip/Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP, Parkin, and ubiquitin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; **p=0.0070).

Data information: For analysis in (C) and (G-J), the number of GFP-positive puncta per cell ranged between 15-111 for WT Tollip, 14-56 for R78A Tollip and 0-45 for CUE mutant Tollip. Statistical significance was determined using a Two-way ANOVA (Dunnett). Bar indicates the mean and error bars represent SEM. Images were captured by widefield immunofluorescence microscopy at 100X magnification. Scale bar represents 10 μM. Zoom insets represent 3.25x magnification in (A) and 4.5x magnification in (B, D, E, and F).
Tollip coordinates Parkin-dependent trafficking of mitochondria-derived vesicles (2020)
Figure 3

Figure 3: Tollip and Parkin are in complex in a CUE domain-dependent manner, but independent of autophagy and Tom1.

(A) HeLa cells expressing mycBioID-Tollip and HA-Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 hours in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS-PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID-Tollip were used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions.

(B) HeLa cells expressing mycBioID alone and HA-Parkin were used to confirm the specificity of the Tollip-Parkin interaction.

(C) Hela cells co-expressing GFP-Tollip and HA-Parkin were either left untreated or treated with AO for 2 hours prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP.

(D) HeLa cells expressing mycBioID-Tollip containing the CUE domain mutation (CUEmut) and HA-Parkin were subjected to biotinylation and streptavidin pulldowns, followed by western blot analysis.

(E) HeLa wild-type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID-Tollip and HA-Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins.
Tollip coordinates Parkin-dependent trafficking of mitochondria-derived vesicles (2020)
Figure 4

Figure 4: Parkin facilitates MDV trafficking and requires an intact UBL domain for Tollip association.

(A) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours, then fixed and stained with antibodies specific to TOM20 (green) and PDH E2/E3bp (red). Nuclei are labelled with Hoechst (blue). Zoom insets represent 5x magnification and arrowheads indicate TOM20-positive MDVs. Scale bar represents 10 µm.

(B) Knockdowns were confirmed for both Parkin and Tom1 by western blotting.

(C, D) Quantification of MDVs, defined as TOM20+ve/PDH-ve, (C) or PDH+ve/TOM20-ve (D) per cell under steady-state conditions were performed (n = 32-52 cells per condition from 3 independent experiments). Statistical significance was determined using a Two-way ANOVA (Dunnett) (ns=not significant). Centre line indicates mean and error bars represent SEM.

(E) Western blot analysis of lysates harvested from SH-SY5Y wild-type (empty vector) and CRISPR generated Parkin KO cells. Immunoblot analysis was performed using antibodies against Parkin and GAPDH.

(F) Immunofluorescence microscopy was performed on SH-SY5Y wild-type and Parkin KO cells either untreated or treated with AO for 2 hours. Cells were immunostained for TOM20 (red), PDH E2/E3bp (blue), and LAMP1 (green). Zoom insets represent 5x magnification. Circles indicate TOM20 MDVs positive for LAMP1 and arrowheads indicate TOM20 MDVs negative for LAMP1. Scale bar represents 10 µm.

(G) Quantitation of TOM20-positive MDV colocalisation with LAMP1 were performed across all treatment groups and represented as the % of TOM20-positive MDVs colocalising with LAMP1. Results represent 3 independent experiments (28-53 cells/condition) and statistical significance was determined using a Two-way ANOVA (Sidak). Centre line indicates mean and error bars represent SEM.

(H) HeLa cells transiently transfected with HA-Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A (AA) for 2 hours, then fixed and stained with antibodies specific to HA, PDH and TOM20. TOM20+ve/PDH-ve MDVs were quantified in cells that were either expressing or not expressing HA-Parkin (n = 12-14 cells per condition from 3 independent experiments). Statistical significance was determined using a Two-way ANOVA (Sidak). Centre line indicates the mean and error bars represent SEM.

(I) Hela cells stably coexpressing BioID-Tollip and either HA-Parkin WT or Δ1-76 (ΔUBL) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns. Western blot analysis was performed on lysate inputs and streptavidin pulldowns (Streptavidin PD) using antibodies against the indicated proteins or epitope tags.
Tollip coordinates Parkin-dependent trafficking of mitochondria-derived vesicles (2020)
Figure 5

Figure 5: Tollip facilitates endosomal 'capture' of MDVs.

(A-B) siRNA knockdown of Tollip or Parkin in SH-SY5Y cells was performed over a 72 hour period, then 18 hours prior to treatment cells were transfected with GFP-2xFYVE to label endocytic membranes. Cells were treated with 25 μM antimycin A (AA) for 2 hours then fixed and immunostained with antibodies specific to PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs that colocalise with GFP-2xFYVE and arrows denote TOM20+ve/PDH-ve MDVs that do not. Zoom insets represent 4.5x magnification.

(C) Quantification of TOM20+ve/PDH-ve MDVs that are positive for GFP-2xFYVE are shown as a % of TOM20 MDVs positive for GFP, with each data point indicating 1 cell. MDV colocalisation were quantified by eye from images captured (n = 20-22 cells per condition from 3 independent experiments). Statistical significance was determined using a Two-way ANOVA (Dunnett). Centre line indicates the mean and error bars represent SEM.

(D) SH-SY5Y cells were left untreated or treated with 25 μM AA for 2 hours prior to fixation and immunostaining for EEA1 (green), PDH E2/E3bp (red), and TOM20 (blue). Arrowheads indicate TOM20+ve/PDH-ve MDVs positive for the early endosome marker EEA1. Zoom insets represent 5x magnification.

(E) siRNA knockdown of Parkin was performed in SH-SY5Y cells followed by 25 μM AA for 2 hours prior to fixation and immunostaining with antibodies specific to TOM20 (green), Tollip (red) and Cytochrome c (blue). We observed Tollip colocalisation with TOM20+ve/PDH-ve MDVs (denoted by arrowheads), which was still maintained following Parkin siRNA knockdown. Zoom insets represent 5x magnification.

(F) SH-SY5Y cells were transfected with GFP-Tollip and treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP (green), Rab7a (red) and PDH E2/E3bp (blue). Zoom insets represent 4x magnification.

(G) Quantification of GFP-Tollip/Rab7a colocalisation using Pearson's correlation. Each data point represents 1 cell (12-14 cells/condition from 3 independent experiments) and bar indicates the mean. Statistical significance was assessed using a One-way ANOVA (Dunnett). Error bars represent SEM.

Data information: Scale bars represent 10 μm.
Tollip coordinates Parkin-dependent trafficking of mitochondria-derived vesicles (2020)
Figure 6

Figure 6: Tollip translocates to a LAMP1 compartment during mitochondrial stress.

(A) Quantitation of Tollip vesicular clusters in SH-SY5Ys treated with 5 μM antimycin A/10 μM oligomycin B (AO) for 2 hours. The percentage of cells with vesicular clusters were quantified from 7-23 cells for each condition from 4 independent experiments. Statistical significance was assessed using an unpaired t-test. Bars indicate the mean value and error bars represent SEM.

(B) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours. The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments. Statistical significance was determined using a Two-way ANOVA (Sidak). Bars indicate the mean value and error bars represent SEM.

(C) Endogenous Tollip and LAMP1 colocalise in SH-SY5Y cells treated with AO for 2 hours. Antibodies specific to LAMP1 (green) and Tollip (red) were used. Zoom insets represent 2.5x magnification.

(D-E) SH-SY5Y cells were transfected with either wild-type GFP-Tollip (WT), GFP-Tollip lacking its N-terminus (∆Nterm), or GFP-Tollip containing a CUE domain mutation (CUEmut) then treated with AO for 2 hours. Cells were fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red). Zoom insets represent 2.5x magnification.

(F) The extent of colocalisation between GFP-Tollip and LAMP1 from untreated and AO treated SH-SY5Y cells were quantitated from immunofluorescence images and represented as a Pearson's correlation (2-3 cells per experiment, from 2 independent experiments). Statistical significance was determined using an unpaired t-test. Bars indicate the mean and error bars represent the SEM.

(G-I) The extent of colocalisation between GFP-Tollip and LAMP1 in Tom1 KO (G), ATG5 KO (H), and VPS35 KO (I) compared to wild-type (WT) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as a Pearson's correlation (2-3 cells per experiment, from 3 independent experiments). Statistical significance was determined using a Two-way ANOVA (Sidak). Bars indicate the mean and error bars represent SEM. Western blot analysis of lysates harvested from wild-type (WT) alongside either Tom1 KO (G) or VPS35 KO (I) Hela cells was performed using antibodies specific to the indicated proteins.

Data information: Images were captured at 100x magnification. Scale bars represent 10 μm.
Tollip coordinates Parkin-dependent trafficking of mitochondria-derived vesicles (2020)
Figure 7

Figure 7: Trafficking of MDVs to the lysosome is dependent on Tollip.

(A) A subset of TOM20+ve/PDH-ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH-SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM antimycin A (AA) for 2 hours. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3bp (red) and TOM20 (blue). Arrows denote TOM20+ve/PDH-ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. Zoom insets represent 4x magnification.

(B) From immunofluorescence images, the total number of TOM20+ve/PDH-ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell (n = 25-32 cells per condition from 3 independent experiments). Each data point represents 1 cell and centre line indicates the mean. Statistical significance was determined using a Two-way ANOVA (Dunnett). Error bars represent the SEM.

(C) siRNA knockdown was performed in SH-SY5Y cells, then 18 hours prior to fixation cells were transfected with GFP-Tollip wild-type (WT), ∆Nterm, CUEmut, or R78A. Cells were fixed and immunostained for PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs. Zoom insets represent 3.5x magnification.

(D) Quantification of TOM20 MDVs per cell was performed by eye (n = 11-14 cells per mutant from 2 independent experiments). Cells expressing a GFP-Tollip construct were identified using the GFP channel. Surrounding cells not expressing GFP were counted as the Tollip siRNA only population. Statistical significance was determined using a one-way ANOVA (Tukey). Each data point represents 1 cell, centre line indicates the mean, and error bars represent SEM.

Data information: Images were captured at 100x magnification. Scale bars represent 10 μm.
A Complex genomic locus drives mtDNA replicase POLG expression to its Disease-Related Nervous System Regions (2017)
Figure 1

Figure 1. Three distant enhancers drive POLG expression in central nervous system.

(left) Luciferase expression in HEK293 cells driven by deletion constructs of POLG proximal promoter. (right) Luciferase expression with mutated CAAT-boxes in POLG promoter. The error bars indicate standard deviation in 3 biological replicates. AU; arbitrary units.

POLG promoter sequence showing predicted CAAT-boxes. Red shows disrupted nucleotides by site-directed mutagenesis in (a).

Expression pattern driven by 500 bp Polg proximal promoter in E12.5 mouse embryo. LacZ-positive cell populations in the developing midbrain (black arrows), dorsal root ganglia (grey arrows) and motoneuron progenitors (arrowhead) of the neural tube. Somites show some expression (white arrow). Sectioning planes indicated by red lines. Scale bars 100 µm.

Prediction of enhancers in the genomic loci of mtDNA maintenance genes, 100 kb upstream of the analyzed gene, found in human-mouse and human-rat comparisons. EEL score for individual elements: red bars. Protein coding genes upstream from mtDNA maintenance genes are shown with black lines under each locus (picture not in scale). POLG shows three highly conserved elements in a gene-poor region. TWNK shows one distant element, with several genes between the element and the gene, suggesting the element not be a specific regulator for TWNK. Abbreviations: TWNK, Twinkle mtDNA helicase; POLG, DNA polymerase gamma, catalytic subunit; POLG2, DNA polymerase gamma, accessory subunit; SSBP1, single stranded DNA binding protein 1; TFAM, mitochondrial transcription factor A.

- (G) POLG enhancer elements are functional in vivo and drive expression in E12.5 transgenic mice. Sectioning planes indicated by red lines. Black line in (E) marks the expression in the dorsal neural tube, whereas rostral and caudal regions lack dorsal expression (black arrows).

(H)- (J) Neural tube lacZ expression driven by (H) EE1: immature neuronal precursors (grey arrow), (I) EE2: dorsal neural tube (grey arrow) and dorsal root ganglia (black arrow), and (J) EE3: dorsal neural tube (grey arrow). Scale bars 100 µm.

(K)- (M) Midbrain lacZ expression, driven by (K) EE1, (L) EE2 and (M) EE3. Black arrow indicates neuronal population from EE1 embryo stained in (N). Scale bars 100 µm.

(N) EE1 drives expression in oculomotor complex; immunofluorescent co-staining with antibodies against ISL1/2 (motoneurons of oculomotor complex; red) and β-Gal (green). LacZ-staining of the region in (k); black arrow. Scale bars 50 µm.
A Complex genomic locus drives mtDNA replicase POLG expression to its Disease-Related Nervous System Regions (2017)
Figure 2

Figure 2. Polg Enhancer EE2 drives expression in adult brain, and EE2 and 3 in sensory interneurons of the adult spinal cord.

Sagittal section of adult mouse brain showing lacZ expression driven by EE2. HC, hippocampus.

EE2-driven expression in rostral migratory stream (black arrow) and in subventricular zone (white arrow). Scale bar 760 µm.

Adult mouse spinal cord, EE2 and (D) EE3 expression pattern. Dorsal horns, laminae I-III (black arrows) and central canal (arrowheads). LacZ staining. Dashed lines in (C) indicates the region of dorsal horn shown in (D), (F) and (G). Inlets in (C) and (E) show POLG immunohistochemistry of dorsal horn and central canal (grey arrow), respectively. Scale bars: 100 µm if not indicated otherwise.

(F)-(G) Calbindin (red) and β-Gal (green) expression in interneurons of the dorsal horn laminae I-III of (F) EE2 and (G) EE3 transgenic adult mice. Immunofluorescence. Scale bars 20 µm.
FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria (2016)
Figure 1

Figure 1. FATE1 is associated to mitochondria in human ACC cells.

A. SF-1, FATE1 and β-tubulin protein levels shown in basal condition and after Dox treatment in H295R/TR, H295R/TR SF-1, H295R/TR FATE1 and H295R/TR N-Flag FATE1 cells.

B. FATE1 mRNA expression is increased in H295R/TR SF-1 cells by Dox treatment. The efficiency of FATE1 knockdown by specific (siFATE1) vs. control (siC) siRNA nucleofection is shown (mean ± SEM; n=4 with 3 replicates/experiment).

C. Immunofluorescence showing endogenous FATE1 protein (red) induction of expression by Dox-treatment of H295R/TR SF-1 cells. SF-1 (green), DAPI (blue). Scale bars, 10 μm.

D. Subcellular localization of endogenous FATE1 (red) and transfected fluorescent markers for Golgi, ER and mitochondria, respectively (green) in Dox-treated H295R/TR SF-1 cells. DAPI (blue). Scale bars, 10 μm.

E. Subcellular localization of the endogenous FATE1 protein (green) in Dox-treated H295R/TR SF-1 cells costained with an antibody against the mitochondrial marker HSP60 (red). SF-1 (blue). Scale bar, 10 μm.

F. Immunogold electron microscopy showing association of FATE1 with the mitochondrial outer surface in Dox-treated H295R/TR N-Flag FATE1 cells. Scale bar, 500 nm.

G. Dox-treated H295R/TR N-Flag FATE1 cells were fractioned into nuclear (N), heavy membranes (HM), light membranes (LM) and cytosolic (C) fractions and localization of SF-1, FATE1, ribosomal protein RPL7, VDAC1 and GAPDH was revealed by immunoblot.

H. Effect of increasing concentrations (0, 1, 10 and 100 μg/ml) of proteinase K and 0.1% Triton X-100 (TX-100) treatment of the mitochondrial fraction from Dox-treated H295R/TR N-Flag FATE1 cells on VDAC1, FATE1, TOM20 and cytochrome c.

I. FATE1 and VDAC1 are associated with the pellet (membrane; P) fraction after high-speed centrifugation of the alkaline-extracted mitochondrial fraction of Dox-treated H295R/TR SF-1 cells, while cytochrome c is found in the supernatant (S).