EMBO/soda-nel huggingface dataset:
Multistability and dynamic transitions of intracellular Min protein patterns (2017)
Figure 5

Figure 5. The effect of cell-shape change during growth on the stability of Min protein patterns.

A. Cytosolic fluorescence during growth of a cell from a small elliptical form into a large rectangular shape. Numbers in red indicate time in hours. Illustrations show the positions and orientations of the cell in the first and last time frames. Green and blue lines indicate the maximum and minimum Feret diameters, respectively.

B. sfGFP-MinD patterns during the growth of the cell shown in A. Illustrations indicate the cell boundaries and oscillation angles observed in the first and last frames (not to scale).

C. Quantitative data obtained from the cell shown in A and B. The maximum and minimum Feret diameters (green and blue), and the measured MinD oscillations (red) were expressed in terms of length (top) and angle (bottom) and plotted against time. The number of cells that fit this category was 41/97. Arrows indicate the time when cell width reached the chamber width of 5 μm.

D-F. Data are presented as in A-C for another cell that showed persistent oscillations along the horizontal axis throughout growth. The number of cells that fit this category was 28/97.

G. Time-lapse images of sfGFP-MinD that reveal stochastic switching of patterns in a cell with an asymmetric shape and a low aspect ratio. White arrows indicate the oscillation axes.

H. The angles of the maximum and minimum Feret diameters (green and blue), and the measured MinD clusters (red) for the cell shown in panel G plotted against time. The number of cells that fit this category was 10/97. All scale bars correspond to 5 μm.
Multistability and dynamic transitions of intracellular Min protein patterns (2017)
Figure 6

Figure 6. Transitions between various modes of Min protein patterns.

A. Time-lapse images showing the transition from a longitudinal pole-to-pole mode to the transverse mode. Scale bar, 5 μm.

B. Time-lapse images showing the transition from a transverse mode to a longitudinal pole-to-pole mode.

C. Bar plot showing the distribution of the number of transitions. Inset: Data from experiments carried out under nutrient-poor conditions in which growth rates are reduced.

D. Representative time-course of a change in the mode of sfGFP-MinD oscillation. The black line is a sigmoidal fit. The dashed black lines indicate 15° and 75° and the dashed red line indicates 45°.

E. Bar plot showing the time scale of the switch in the oscillations. Inset: Data from experiments carried out in nutrient-poor conditions.

F. Bar plots showing the relative numbers of the indicated transitions that occur at different cell lengths. All cells have a width of 5 μm.

G. Distribution of final patterns in cells of the indicated widths as indicates, and lengths of 9-10 μm.

H. Time-lapse images of various modes of transitions between patterns. Cell sizes from top to bottom are respectively 10x2x1, 10x6x1, 9x5x1, 10x4x1 μm3. Note that the cells are scaled differently. On the right is an illustration showing Min pattern transitions through intermediate states.

Legends for Extended View Figures
YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis (2021)
Figure 1

Figure 1. YAP/TAZ are key drivers of Sorafenib resistance by inhibiting ferroptosis.

(a) Scheme of the shRNA-mediated synthetic lethal screen. Huh7-IR and CR cells were infected with lentiviral vectors (MOI=0.5) expressing the shRNA library (human genome-wide pooled lentiviral shRNA library module 1, Vector: pRSI16cb, Cellecta) and cultured with 2μg/ml Puromycin for the selection of shRNA-expressing cells plus 7μM Sorafenib (Srf). After 4 weeks of culture, genomic DNA was extracted, and shRNA barcodes were amplified for next-generation sequencing to uncover the critical genes for Sorafenib resistance.

(b) Combinatorial analysis of genes differentially expressed between Sorafenib-sensitive and resistant cells and genes with depleted barcodes in the synthetic lethal screen in Huh7-IR and CR cells. 38 common genes were identified, among which was WWTR1 coding for TAZ.

(c) Huh7-parental, IR and CR, and Hep3B-parental, and IR and CR cells were treated with different concentrations of Sorafenib (0, 3, 6, 9μM) for Huh7-P/IR/CR and Sorafenib (0, 2, 4, 6μM) for Hep3B-P/IR/CR for 18 hours before harvest. Protein levels of YAP and TAZ were determined by immunoblotting illustrating higher protein levels of YAP/TAZ in Sorafenib-resistant cells. GAPDH served as loading control. Results represent three independent experiments.

(d) Colony formation assay showing that shRNA-mediated depletion of YAP/TAZ leads to cell numbers in response to Sorafenib treatment. Huh7 IR and CR cells either expressing a control shRNA (shLuc, non-targeting shRNA) or shRNA against both YAP and TAZ (shY/T) were treated with different concentrations of Sorafenib (0, 4, 8μM) for 2 weeks and colonies were visualized by crystal violet staining. Results represent three independent experiments.

(e) Gene Set Enrichment Analysis (GSEA) of the genes differentially expressed between YAP/TAZ-deficient (siY/T) and control siRNA (siCtrl) transfected HLE cells showed an enrichment for genes involved in the regulation of lipid peroxidation.

(f) Basal reactive oxygen (ROS) levels increased upon loss of YAP/TAZ. HLE-shLuc and HLE-shY/T cell lines were stained with CellROX™ Green Flow Cytometry Assay Kit, and ROS levels were measured by flow cytometry using a 488 nm laser. Results represent three independent experiments.

(g) Basal lipid peroxidation levels increased with the loss of function of YAP/TAZ. HLE-shLuc and HLE-shY/T cells were stained with C11-BODIPY 581/591. Reduced-Bodipy was measured by flow cytometry using a 488 nm laser, and oxidized-Bodipy was measured with a 561 nm laser. A significant shift of oxidized-Bodipy occurred upon depletion of YAP/TAZ. Results represent three independent experiments.

(h) Colony formation assay demonstrating that the ferroptosis inhibitor Ferrostatin-1 (Fer) reversed Sorafenib-induced cell death in YAP/TAZ-deficient HCC cells. HLE-shLuc and shY/T cells were treated with different concentrations of Sorafenib (0, 2, 4μM) and either DMSO or Ferrostatin-1 (Fer; 5μM) for 2 weeks. Results represent three independent experiments.
YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis (2021)
Figure 6

Figure 6. Targeting ferroptosis overcomes Sorafenib resistance.

(a) Establishment of a YAP/TAZ-deficient SNU398 cell line. SNU398 cells were infected with lentivirus to stably express either shLuc as control or shYAP/TAZ. Immunoblotting illustrated the loss of YAP/TAZ expression. GAPDH served as loading control.

(b - d) Combination of YAP/TAZ deficiency and Sorafenib treatment suppressed tumor growth in a HCC xenograft model. SNU398-shLuc or SNU398-shYAP/TAZ (shY/T) cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with 20mg/kg Sorafenib or vehicle control, and tumor sizes were measured twice a week (b). Tumor weights and sizes were also recorded after sacrifice of the mice (c,d). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers of shLuc + Vehicle, shY/T + Vechicle and shY/T + Srf were 4 per cohort, mouse numbers of shLuc + Srf were 3 per cohort.

(e) Pharmacological inhibition of glutathione synthesis and function sensitizes HCC tumors to Sorafenib therapy. SNU398 cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with vehicle control or with 20mg/kg Sorafenib alone, or with 20mg/kg Sorafenib and 20mM BSO in the drinking water or 120mg/kg SSA per os. Tumor sizes were measured twice a week. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers were 8 for the experimental cohorts Vehicle, Srf, and Srf + SSA and 7 for Srf + BSO.

(f) Representative pictures of immunohistochemical staining of Ki67 and 4-HNE in tissue sections taken from HCC tumors of the mice treated as described in (e). Ki67 is a marker for cell proliferation, while 4-HNE is a marker of lipid peroxidation and ferroptosis. Scale bars, 50μm.

(g) Quantification of 4-HNE-positive cells of the immunohistochemical stainings described in (f). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Each data point represents the average of 3 technical replicates of one histological section, the numbers of tumor samples are given in (e).
Cooperative Amyloid Fibre Binding and Disassembly by the Hsp70 disaggregase (2022)
Figure 4

Figure 4. Hsc70 recruitment is greatly stimulated by Apg2.

(A) Binding assay for Hsc70 binding in the presence of WT DNAJB1: control of DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70/Apg2, control of DNAJB1/Hsc70/Apg2. The '*' highlights the condition where an increase of Hsc70 in the bound fraction is observed. P, pellet; S, supernatant. The amount of Hsc70 recruited to the fibres is shown in the pellet fractions.

(B) Binding assay for Hsc70 binding in the presence of the truncated ΔJ-DNAJB1, lacking the J domain. The gel layout is similar to the previous one with ΔJ-DNAJB1 replacing WT DNAJB1. Both Apg2 and J domain are required for enhancement of Hsc70 binding.

(C) Histogram of Hsc70 bound fraction in each sample (N = 3 independent experiments, WT: WT DNAJB1, ΔJ: ΔJ-DNAJB1 mutant, -Apg2: condition with fibres + DNAJB1/Hsc70, +Apg2: condition with fibres + DNAJB1/Hsc70/Apg2). A 2-way ANOVA was performed with Tukey's multiple comparisons test (**: P=0.003; ****: P<0.0001). Mean Hsc70 binding values with SD are shown.

(D) TIRF microscopy images of the labelled αSyn fibres and Hsc70 in the absence or in the presence of Apg2. Scale bar, 1 µm.

(E, F) Quantitation of fluorescence intensity of Hsc70 (E) and αSyn (F) in the two conditions (+/- Apg2, N = 3 independent experiments, n = 65 fibres). Mean Hsc70 and αSyn fluorescence intensities with SD are shown.

(G) Quantitation of differences in Hsc70 binding between the two ends of the fibres, normalised to the local αSyn intensity, in the absence and presence of Apg2 (N = 3 independent experiments, n = 54 fibres). Mean Hsc70 fluorescence intensities with SD are shown.

Data information: (E-G) For each plot, data were tested for normality by performing a Shapiro-Wilk test. As the data did not display a normal distribution, they were analysed using a two-tailed Mann-Whitney test (****: P<0.0001, ns: not significant).
Cooperative Amyloid Fibre Binding and Disassembly by the Hsp70 disaggregase (2022)
Figure 5

Figure 5. Dependence of Hsc70 recruitment and disaggregation activity on Apg2 concentration.

Binding assay with different dilutions of Apg2 incubated with DNAJB1/Hsc70 and (A) with αSyn fibres or (B) without αSyn fibres (control). 5 mM ATP was present in the buffer throughout.

(C) Plot showing Hsc70 binding as a function of Apg2:Hsc70 molar ratio (N = 3 independent experiments). The action of Apg2 in recruiting Hsc70 to the fibres saturates at a molar ratio of Apg2:Hsc70 of 1:10. The curve indicates the mean Hsc70 binding values.

(D) Disaggregation activity measured by Thioflavin T fluorescence shows the same dependence on Apg2 as binding assays. High Apg2 (1:2 molar ratio) makes disaggregation less efficient, possibly by prematurely releasing Hsc70.

(E) Quantification of the disaggregation efficiency as a function of Apg2:Hsc70 molar ratio (N = 3 independent experiments). The disaggregation efficiency was determined by taking the values from the final hour of the assay curves in (D). The curve indicates the mean efficiency values.

(F), Plot showing the linear correlation between disaggregation activity and Hsc70 binding. Including the data point without Apg2 did not change the correlation coefficient, which remained at 0.967). In contrast, including the highest Apg2 concentration (Apg2:Hsc70 molar ratio of 1:2) reduced the correlation coefficient to 0.904. Mean values with SD are shown.
Cancer-associated POT1 mutations lead to telomere elongation without inducing a DNA damage response (2021)
Figure 1

Figure 1: The Q623H caPOT1 mutation does not induce a DNA damage response at telomeres

Schematic of the POT1 locus and editing strategy used to engineer hESCs with the Y223C and Q623H caPOT1 mutations.

Representative images for the TIF analysis shown in (C). Shown are POT1Y223C/- hESCs and an unedited wild-type hESC clone (WT/WT) isolated in parallel, as well as a POT1 WT/Q623H hESC and a contemporaneously isolated hECS clones (WT/WT). Scale bars = 10µm.

Box and whisker plot (showing min, max and average) quantifying telomere-dysfunction induced foci (TIF) in two POT1 Y223C/- hESC lines, one POT1 Q623H/Q623H, and one POT1WT/Q623H hESC lines compared to wild-type unedited control hESCs. Cells were analyzed 3 months post targeting. Cells were stained for TRF1 and γ-H2Ax. POT1Y223C/- cells have increased TIFs compared to an unedited wild-type cell clone, isolated and cultured in parallel (P<0.01, two-tailed Mann-Whitney test, one biological replicate).

Images of telomere and centromere FISH on metaphase spreads of cell lines as in (B). Cy3 Tel-C PNA probes were used. Arrows indicate chromosomal abnormalities.

E) Quantification of chromosomal abnormalities as determined by FISH analysis in (D).
Cancer-associated POT1 mutations lead to telomere elongation without inducing a DNA damage response (2021)
Figure 3

Figure 3: A heterozygous Y223C caPOT1 mutation does not induce a DNA damage response at telomeres, but elongates telomeres

A) Schematic for the "scarless loopout" strategy used to generate POT1WT/Y223C in hESCs.

B) TIF analysis of wild-type, POT1WT/Y223C hESCs, and the indicated controls using antibodies against TRF1 and γ-H2AX. Scale bars = 10µm. Lentiviral expression of TPP1∆PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response. TPP1∆PBD infected cells were analyzed 72h post infection and without selection.

C) Box and whisker plot (showing min, max and average) quantifying the fraction of TIF-positive telomeres per cells as shown in (B) (two-tailed Mann-Whitney test, one biological replicate).

D) STELA showing telomeres of POT1WT/Y223C, POT1WT/Puro, and an untargeted control hESC clone that was isolated and cultured in parallel to the edited clone.

E) Quantification of two STELA experiments, including that shown in (D). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.
Cancer-associated POT1 mutations lead to telomere elongation without inducing a DNA damage response (2021)
Figure 5

Figure 5: POT1 mutations are not selected against in vivo

A) Experimental outline for the xenografting of CAS9-edited hCD34+ cells into mice, isolation of human cells from the bone marrow, and sequential in vitro differentiation.

B) Editing outcomes of hCD34+ cells nucleofected with POT1 sgRNA-1 before transplantation determined by NGS.

C) Representative genotyping outcomes of hCD34+ cells nucleofected sgRNA-1, xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS.

Data information: (B, C) The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion. A dash inside a gray box indicates a deleted base pair.
ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis (2022)
Figure 1

Figure 1. Enkd1 knockout mice display ciliogenesis defects in multiple organs.

(A) Strategy for the generation of Enkd1 knockout mice. Deletion of a fragment containing the 173-bp exon 3 and adjacent flanking sequences results in the disruption of ENKD1 protein expression.

(B) Identification of wild-type (WT), heterozygous (HZ), and knockout (KO) mice by PCR and agarose gel electrophoresis.

(C) Immunoblotting (IB) of ENKD1 and α-tubulin in wild-type and Enkd1 knockout mice.

(D-F) Immunofluorescence images (D) and quantification of the density of cilia (E, n = 20 fields from three mice) and ciliary length (F, n = 100 cilia from three mice) in mouse retinas stained with antibodies against acetylated α-tubulin (Ace-α-tubulin) and γ-tubulin and DAPI. Scale bar, 2 µm.

(G-H) ERG recording images (G) and a-b wave amplitudes (H, n = 5 mice) show the difference in the ERG a-b wave amplitude between wild-type and Enkd1 knockout mice.

(I-J) VEP recording images (I) and N2-P2 wave amplitudes (J, n = 5 mice) show the difference in the VEP N2-P2 wave amplitude between wild-type and Enkd1 knockout mice.

(K-M) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 mice) and ciliary length (M, n = 100 cilia from three mice) in mouse kidneys stained with the antibody against acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (L), >200 cells from 12 images were analyzed for each mouse. Scale bar, 3 µm.

(N-Q) Images (N) and quantification of the percentage of flagellated sperm (O, n = 9 mice), flagellar length (P, n = 100 sperm from nine mice), and the percentage of sperm with normal motility among flagellated sperm (Q, n = 9 mice) for wild-type and Enkd1 knockout mice. To quantify the percentage of flagellated sperm (O), >200 sperm were analyzed for each mouse. To quantify the percentage of sperm with normal motility among flagellated sperm (Q), >200 flagellated sperm were analyzed for each mouse. In panel N, the arrows indicate two spermatozoa without tails. Scale bar, 50 µm.

(R, S) Scanning electron microscopy images of cilia (R) and quantification of the percentage of ciliated cells (S, n = 9 mice) in the mouse tracheal epithelium. To quantify the percentage of ciliated cells (S), >200 cells from six fields were analyzed for each mouse. Scale bar, 5 µm.

Data information: Data are presented as mean ± SEM. Unpaired two-tailed t-test was performed. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), ns, not significant.
ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis (2022)
Figure 2

Figure 2. ENKD1 promotes the initiation of ciliogenesis in cultured cells.

(A) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum-free medium for 48 h.

(B-D) Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 70 cilia from three independent experiments) for MEFs cultured in serum-free medium and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (C), >100 cells were analyzed for each experiment. Scale bar, 5 µm.

(E) Immunoblot analysis of ENKD1 and β-actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum-free medium for 48 h.

(F-H) Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) and ciliary length (H, n = 100 cilia from three independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (G), >150 cells were analyzed for each experiment. Scale bar, 5 µm.

(I, J) Immunofluorescence images (I) and quantification of the percentage of ciliated cells (J, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against Arl13b and Centrin and DAPI. To quantify the percentage of ciliated cells (J), >80 cells were analyzed for each experiment. Scale bar, 2 µm.

(K, L) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP, GFP-ENKD1, GFP-ENKD1-N, GFP-ENKD1-M, or GFP-ENKD1-C, cultured in serum-free medium, and stained with the antibody against acetylated α-tubulin and DAPI. The siRNA-resistant forms of ENKD1 were used for these rescue experiments. To quantify the percentage of ciliated cells (L), >120 cells were analyzed for each experiment. Scale bar, 3 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two-tailed t-test was performed. p < 0.01 (**), p < 0.001 (***), ns, not significant.
ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis (2022)
Figure 3

Figure 3. ENKD1 is a component of the centrosome.

(A) Immunofluorescence images of RPE1 cells cultured in normal serum medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm.

(B) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 1 µm.

(C) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 2 µm.

(D-F) 3D-SIM super-resolution images (D), quantification of the outer toroid diameters (E, n = 20 cells from three independent experiments), and a model illustrating the relative positions of γ-tubulin, Ninein, ENKD1, CEP164, CEP912, and Centrin (F) within a centriole. RPE1 cells were serum-starved and stained with antibodies against the indicated proteins. Scale bar, 1 µm.

(G) Immunofluorescence images of RPE1 cells cultured in normal serum or serum-free medium, and stained with antibodies against ENKD1 and γ-tubulin and DAPI. Scale bar, 1 µm.

(H) Immunofluorescence images of RPE1 cells cultured in serum-free medium, and stained with antibodies against ENKD1 and acetylated α-tubulin and DAPI. Scale bar, 1 µm.

(I) Immunofluorescence images of RPE1 cells transfected with GFP-ENKD1 or GFP vector, cultured in a serum-starved condition, and stained with the antibody against acetylated α-tubulin and DAPI. Scale bar, 1 µm.

(J) Schematic diagrams showing the centrosomal localization domain of ENKD1.

(K) 3D-SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum-free medium (0, 12, 24, or 48 h of serum starvation), and stained with antibodies against ENKD1 and acetylated α-tubulin. Scale bar, 1 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SD.