EMBO/soda-nel huggingface dataset:
THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer (2021)
Figure 2

Figure 2: Loss of THEM6 alters lipid homeostasis.

A, Heatmap illustrating the steady-state levels of significantly regulated lipids in THEM6 KO 22rv1 cells when compared to CTL (p ≤ 0.05, FC = 1.3). Values are expressed as log2(FC).

B-C, Changes in lipid content (total amount) observed in THEM6 KO CRPC cells when compared to CTL.

D, Heatmap illustrating the steady-state levels of significantly regulated lipids in THEM6 OE CWR22res cells when compared to EV (p ≤ 0.05, FC = 1.2). Values are expressed as log2(FC).

E, Changes in lipid content (total amount) observed in THEM6 OE CWR22res cells when compared to EV.

F, Average Raman spectra of CWR22res CTL and THEM6 KO orthografts.

G-H, Quantification of tumour lipid (2845 cm-1-peak) and cholesterol (2880 cm-1-peak) content obtained from F.

Data information: AU = arbitrary unit. Panels B, C, E : Data are presented as mean values +/- SD. Panels G, H: Centre line corresponds to median of data, top and bottom lines correspond to upper and lower quartiles. Statistical analysis: B, C: *p-value < 0.05 using a 1-way ANOVA with a Dunnett's multiple comparisons test. E: *p-value < 0.05 using a two-tailed Student's t-test. G, H: Kruskal-Wallis test. Data reproducibility: Panels A, B, C, D, E: n = 3 independent biological experiments. Panel F: n = 4 mice per group. Panel G, H: n = 6581 (CTL); 12047 (KO1); 6493 (KO2) peak intensities that were extracted from 4 mice per group. EV: Empty Vector; DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester.
THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer (2021)
Figure 3

Figure 3: THEM6 interacts with multiple ER membrane components involved in protein transport.

A, Venn diagram highlighting commonly modulated proteins (p-value ≤ 0.05, FC = 1.3) in THEM6 KO 22rv1 cells (2 clones) when compared to CTL. Up-regulated proteins are on top; Down-regulated proteins are into brackets.

B, Enriched cellular components commonly down-regulated in THEM6 KO 22rv1 cells (2 clones) when compared to CTL.

C, Representative electron microscopy (EM) pictures of CTL and THEM6 KO 22rv1 cells taken at low (left) and high (right) magnification. Red arrows point towards abnormal ER structure. Scale bar represents 500 nm.

D, Quantification of ER membrane length, plasma membrane length and ER/plasma membrane ratio using the EM pictures from C.

E, Immunofluorescence showing co-localisation of THEM6 and the ER marker calreticulin in 22rv1 cells. Scale bar represents 20 µm.

F, Prediction of transmembrane domain in the sequence of the THEM6 protein. Topology prediction was performed using the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM).

G, Western blot analysis of THEM6 expression in cytoplasmic (cyto.), membrane/organelle (memb.) and nuclear fractions (nucl.) of 22rv1 cells. HSC70 and SP1 were used as cytoplasmic and nuclear-enriched markers, respectively.

H, Enriched cellular components in THEM6-interacting proteins following THEM6 pulldown in T6 OE HEK293 cells.

I, Western blot analysis of THEM6, AMFR, SEC61b and XPO1 expression in CRPC cells following THEM6 immunoprecipitation in CRPC cells.

J, Western blot analysis of CALX and CALR expression in PCa cells following THEM6 silencing.

Data information: Panels B, H: Enrichment analysis were performed using the STRING database (http://string-db.org). Panel D: Data are presented as mean values +/- SD. Statistical analysis: D: Unpaired t-test. Data reproducibility: Panel D: n = 20 cells/condition. Panels E, G, J: representative image from 3 independent biological experiments. Panel I: representative image from 2 independent biological experiments.
THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer (2021)
Figure 4

Figure 4: Loss of THEM6 affects de novo sterol and fatty acid synthesis.

A, Proteomic analysis highlighting proteins associated with sterol biosynthetic pathway and significantly down-regulated in THEM6 KO 22rv1 cells when compared to CTL (p-value ≤ 0.05, FC = 1.3).

B, RT-qPCR analysis of MVD, FDPS and DHCR7 expression in CTL and THEM6 KO 22rv1 cells. CASC3 was used as a normalising control.

C, Gene set enrichment plots analysed from THEM6 KO LNCaP AI cells using the GOBP "Sterol homeostasis" gene set.

D, Labelled desmosterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation.

E, Relative isotopologue distribution of desmosterol in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation.

F, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO CWR22res cells after 72 hours of incubation.

G, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO MCF-7 cells after 72 hours of incubation.

H, Pearson's correlation analysis of SQLE, LSS, DHCR7 and DHCR24 with THEM6 using the PRAD TCGA dataset. Results were obtained using the GEPIA website http://gepia.cancer-pku.cn/.

I, Differential expression of ACACA in high and low THEM6 tumours according to the PRAD TCGA dataset.

J, Labelled palmitic, oleic and stearic acid fractions derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation.

K, Relative isotopologue distribution of palmitic acid in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation.

Data information: Panels A, B, D, E, F, G, J, K: Data are presented as mean values +/- SD. Panel I: Centre line corresponds to median of data, top and bottom of box correspond to 90th and 10th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Statistical analysis: B, D, E, F, J, K: *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. G: *p-value < 0.05 using a two-tailed Student t-test. I: two-tailed Mann-Whitney U test. Data reproducibility: Panels A, B: n = 3 independent biological experiments. Panels D, E, F, G, J, K: n = 3 independent wells from the same cell culture. Panel I: n = 225 tumours for THEM6 low and n = 121 tumours for THEM6 high.
THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer (2021)
Figure 5

Figure 5: THEM6-mediated lipid remodelling is required for UPR activation.

A, Enriched biological processes commonly down-regulated in THEM6 KO 22rv1 cells (2 clones) when compared to CTL.

B, Proteomic analysis highlighting proteins associated with the ER stress response significantly down-regulated in THEM6 KO 22rv1 cells when compared to CTL (p-value ≤ 0.05, FC = 1.3).

C, Western blot analysis of BIP, XBP1s, ATF4, CHOP and THEM6 expression in CTL and THEM6 KO 22rv1 cells.

D, Proliferation of CTL and THEM6 KO CRPC cells treated with tunicamycin (2.5 µg/ml) for 72 hours. Cell count is normalised to initial number of cells at T0.

E, Proteomic analysis highlighting ATF4 targets significantly down-regulated in THEM6 KO LNCaP AI cells when compared to CTL (p-value ≤ 0.05, FC = 1.3).

F, Western blot analysis of ATF4 and THEM6 expression in CTL and THEM6 KO LNCaP AI cells.

G, Western blot analysis of ATF4, CHOP and THEM6 expression in PCa cells following THEM6 silencing.

H, Differential expression of EIF4G1 and ASNS in high and low THEM6 tumours according to the PRAD TCGA dataset.

I, Western blot analysis of ATF4 and CHOP expression in CTL and THEM6 KO 22rv1 cells treated with palmitic acid (200 µM) for 48 hours.

J, Western blot analysis of ATF4 and CHOP expression in CTL and THEM6 KO LNCaP AI cells treated with palmitic acid (200 µM) or hexadecylglycerol (50 µM) for 48 hours.

Data information: Panels C, F, G, I, J: HSC70 was used as a sample loading control. Panels B, D, E: Data are presented as mean values +/- SD. Panel H: Centre line corresponds to median of data, top and bottom of box correspond to 90th and 10th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Statistical analysis: D: 1-way ANOVA with a Dunnett's multiple comparisons test. H: two-tailed Mann-Whitney U test. Data reproducibility: Panels C, F, G, I, J: representative image from 3 independent biological experiments. Panels B, D, E: n = 3 independent biological experiments. Panel H: n = 225 tumours for THEM6 low and n = 121 tumours for THEM6 high.
BNIP3L/NIX regulates both mitophagy and pexophagy (2022)
Figure 1

Figure 1: Iron chelation sequentially induces selective autophagy pathways

A. Representative immunoblots and quantifications (below) of the indicated proteins in lysates of ARPE-19 cells treated with 1 mM DFP for the indicated time (h, hours).

B-C. Representative confocal images of ARPE-19 cells stably expressing the mito-QC reporter (B), or the pexo-QC reporter (C) treated with 1 mM DFP for the indicated time (h, hours). Enlarged images of the area outlined in white are shown in the lower corners. The 'red-only' mask is generated during images analysis and highlights red-only puncta (autolysosomes) based on the mCherry/GFP ratio and thresholding criteria. Nuclei were stained in blue (Hoechst). Scale bar: 10μm. At right, quantification of total red-only punctate per cell (mitolysosomes or pexolysosomes).

D. ARPE-19 cells were transfected with 50 pmol of non-targeting siRNA (siNT) or 50 pmol of siRNA targeting ATG13 or ULK1. 48 hours post knockdown, cells were treated with 1 mM DFP for an additional 48 hours. Cell lysates were subject to immunoblotting with the indicated antibodies. Quantification is shown on the right.

All data are mean ± s.d; Statistical significance is displayed as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P≤ 0.0001; ns, not significant. A, n = 4 biological replicates, one-way ANOVA, Dunnett's multiple comparisons test. B, C, n = 3 biological replicates with >40 cells per replicate, one-way ANOVA, Tukey's multiple comparisons test. D, n = 4 biological replicates, two-way ANOVA, Sidak's multiple comparisons test.
BNIP3L/NIX regulates both mitophagy and pexophagy (2022)
Figure 2

Figure 2: Depletion of NIX restricts both mitophagy and pexophagy

A. Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells, treated with 1mM DFP for 48 hours.

B. Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo-QC reporter and treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).

C. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins in lysates of WT or two NIX KO ARPE-19 clones (Cl), treated with 1 mM DFP for the indicated time (h, hours).

D-E. Representative confocal images of WT or NIX KO ARPE-19 cells stably expressing the mito-QC reporter (D) or the pexo-QC reporter (E) treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).

F. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins. NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe empty vector (∅) or a pBabe NIX vector, were treated with 1 mM DFP for 48 hours prior lysis. Two biological replicates are shown in the immunoblot.

Enlarged images of the area outlined in white are shown in the lower corners. Scale bar: 10μm.

Overall data are mean ± s.d; Statistical significance is displayed as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P≤ 0.0001; ns, not significant. B, n=3 biological replicates; C, n = 3 biological replicates, statistical comparisons were made within the same time point; D-E, n=4 biological replicates; F, n=4 biological replicates; two-way ANOVA, Tukey's or Sidak's multiple comparisons test.
BNIP3L/NIX regulates both mitophagy and pexophagy (2022)
Figure 3

Figure 3: NIX localises to peroxisomes via its transmembrane domain

A-B. Representative confocal images of untreated (CTRL) and DFP-treated ARPE-19 cells (1mM, 48 hours), stably expressing GFP-WT NIX (green) and stained with anti-catalase antibody (A, red) or anti-PMP70 antibody (B, red). At right, quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70, with or without DFP treatment for 48 hours. Peroxisomes enriched for GFP-NIX or having minimal signal for GFP-NIX are depicted with white arrowheads or white arrows, respectively.

C. Representative confocal images of ARPE-19 cells stably expressing GFP-WT NIX, GFP-NIXΔTM or GFP-NIX TM only (green), treated with DFP (1mM) for 48 hours and immunostained with anti-catalase antibody (red). At right, quantification of Pearson correlation coefficients between the green and the red signal.

Overall data are mean ± s.d; Statistical significance is displayed as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P≤ 0.0001; ns, not significant. n = 3 biological replicates with >15 cells per replicate, A-B, unpaired t-test, two tailed; C, one-way ANOVA, Tukey's multiple comparisons test.

D. Representative confocal images of untreated (CTRL) and DFP-treated ARPE-19 cells (1mM, 48 hours), stably expressing GFP- WT NIX (green) and immunostained with a mitochondrial marker (ATPB, cyan) and peroxisomal marker (catalase, red).

Enlarged images of the area outlined in white are shown on the right. Nuclei were stained in blue (Hoechst) and scale bars: 10μm.
BNIP3L/NIX regulates both mitophagy and pexophagy (2022)
Figure 4

Figure 4: NIX is targeted to peroxisomes in the absence of mitochondria

A. Schematic illustration of mitochondria depletion in cells overexpressing Parkin(PRKN). Cells are pre-treated for 24 hours with CCCP (20 µM), and after 1 day of recovery (CCCP washout), treated for 48 hours with 1mM DFP.

B. Representative immunoblots of the indicated proteins in lysates of WT or HA-Parkin(PRKN)- ARPE-19 cells treated as in A.

C-D. Representative confocal images of HA-Parkin (PRKN) ARPE-19 cells treated as in A and stained with anti-NIX antibody (green) and anti-ATPB antibody (red, C) or anti-Catalase antibody (red, D). Enlarged images of the area outlined in white are shown on the right. Nuclei were stained in blue (Hoechst) and scale bars: 10μm.
BNIP3L/NIX regulates both mitophagy and pexophagy (2022)
Figure 5

Figure 5: NIX LIR domain is required for mitophagy and pexophagy

A. Representative confocal images of ARPE-19 cells stably expressing mCherry-SKL (peroxisomes, red), treated with 1mM DFP for 48 hours and 50 nM Bafilomycin A (BafA) for the last 6 hours prior imaging. Mitochondria were stained with anti-COXIV antibody (green) and autophagosomes with anti-LC3 antibody (cyan). Green-only, red-only, and green and red positive structures are depicted with yellow, red and white arrowheads, respectively. Scale bars for i. and ii. is 2μm. Enlarged images of the area outlined in white are shown on the right.

B. Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe Flag vector (∅), a pBabe Flag-NIX vector (NIX), a pBabe Flag-NIX W36A\L39A vector (W36A\L39A) or a pBabe Flag-NIX S34E\S35E vector (S34E\S35E), and treated with 1 mM DFP for 48 hours prior lysis.

C-D. Representative confocal images of cells as in B, stably expressing the mito-QC reporter (C) or the pexo-QC reporter (D) and treated with 1 mM DFP for 24 hours (C) or 48 hours (D) (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel). Enlarged images of the area outlined in white are shown in the lower corners. Nuclei were stained in blue (Hoechst) and scale bars: 10μm.

Overall data are mean ± s.d; Statistical significance is displayed as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P≤ 0.0001; ns, not significant. C, n=3 biological replicates; D, n = 4 biological replicates; one-way ANOVA, Tukey's multiple comparisons test.
BNIP3L/NIX regulates both mitophagy and pexophagy (2022)
Figure 6

Figure 6: Figure 6: NIX KO mouse retinas are enriched in peroxisomes

A. Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers (PMP70 and catalase) or a lysosomal marker LAMP1. Nuclei are coloured in white (DAPI). RPE, retinal pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL; inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cells. Scale bar: 20 mm.

B. Quantification of signal area in the ONL and INL from images as A. Each data point represents a single mouse (n=3-4 per group). Overall data are mean ± s.d; Statistical significance is displayed as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P≤ 0.0001; ns, not significant. Unpaired t-test, two tailed.

EXPANDED VIEW FIGURE LEGENDS
Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex (2019)
Figure 1

Figure 1. Rod is required for Mad1 localization and checkpoint response to unattached kinetochores

A) Schematic of Rod RNAi depletion and synchronization protocol.

B) Representative still images of control, Rod depleted cells and Rod depleted cells supplemented with RNAi resistant Venus-Rod treated with nocodazole and time in minutes indicated. Scale bar, 5 μm.

C) Time from NEBD to mitotic exit for the indicated conditions with each circle representing a single cell analyzed and mean time indicated by red line and standard error of mean shown. The number of cells counted and mean time indicated above. A Mann Whitney test was used for statistical analysis (ns: non-significant, ****: P ≤ 0.00001). A representative result from at least 3 independent experiments is shown.

D) Immunofluorescence analysis of ZW10 and Mad1 upon depletion of Rod. Scale bar, 5 μm.

E) Quantification of kinetochore intensities of the indicated proteins in control or Rod depleted cells with the signal normalized to CREST levels and Bub1 pSpT normalized to Bub1. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.

F) Time from NEBD to mitotic exit in control depleted, Rod depleted, Bub1 depleted and Rod and Bub1 co-depleted HeLa, U2OS and RPE1 cells. Cells were treated with nocodazole. (Mann Whitney test, ns: non-significant, ****: P ≤ 0.00001). A representative result from at least 3 independent experiments is shown.

G) Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells. Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.
Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex (2019)
Figure 2

Figure 2. Penetrant Rod removal by combing CRISPR/Cas9 and RNAi.

A) Western blot analysis of whole cell extract for the indicated conditions. Quantitative analysis of Rod levels using LiCor technology is indicated below. The asterisk indicates an unspecific band.

B) Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions. Kinetochore signal is normalized to CREST signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.

C) Representative still images of unperturbed mitosis for parental cells, Rod C cells and Rod CR cells with time in minutes indicated. CFP-Histone 3 was used here as the chromosome marker. Scale bar, 5 μm.

D, E) Time from NEBD to exit of indicated conditions with cells treated with nocodazole or taxol. Red circles represent cells still arrested in mitosis when the filming stopped. (Mann Whitney test, ns: non-significant, ****: P ≤ 0.00001). Mean (red line) and standard error of mean (black bar) indicated. A representative result from at least 3 independent experiments is shown.
Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex (2019)
Figure 3

Figure 3. Bub1 is required for SAC signaling from unattached kinetochores.

A, B) Time from NEBD to exit in indicated conditions with cells treated with nocodazole or taxol. Red circles represent cells still arrested in mitosis when the filming stopped. (Mann Whitney test, ns: non-significant, *: P ≤ 0.05, ****: P ≤ 0.00001). Mean (red line) and standard error of mean (black bar) indicated. A representative result from at least 3 independent experiments is shown.

C) Mass spectrometry analysis of BubR1 IPs from the indicated cell lines. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions. Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated.

D) Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines. In Bub1 CR we only detected one peptide from three purifications. Standard deviation indicated.

E) Representative still images of unperturbed mitosis for parental cells, Bub1 C cells and Bub1 CR cells with time in minutes indicated. CFP-Histone 3 was used as the chromosome marker. Scale bar, 5 μm.
Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex (2019)
Figure 4

Figure 4. Mad1 localization and kinetochore dynamics

A, B) Tethering of Mad1 (Mad1 485-718) to Ndc80, KNL1 (Zhang et al, 2017) or Bub1 1-553ΔCD1 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant, ****: P ≤ 0.00001). A representative result from at least 3 independent experiments is shown.

C) Localization of Venus-tagged Mad1 in parental HeLa cells, Bub1 CR cells and Rod CR cells in the presence of nocodazole. Scale bar, 5 μm.

D) Still images from FRAP experiments using Venus-tagged Mad1 in the indicated conditions. Image before bleach is shown and then time following bleach. Arrowheads indicate kinetochore pairs. Scale bar, 5 μm for whole cell image and 1 μm for the zoom in on kinetochores.

E) Analysis of Rod levels in cells used for FRAP experiment.

F) Venus-tagged Mad1 kinetochore intensity versus time from bleaching shortly after NEBD in unperturbed cells (left) or in cells arrested with nocodazole (right). Data combined from 3 independent experiments and standard deviation indicated.