EMBO/soda-nel huggingface dataset:
Disrupting metformin adaptation of liver cancer cells by targeting  the TOMM34/ATP5B axis (2022)
Figure 2

Fig. 2. TOMM34 promotes HCC growth and metastasis during metformin adaptation.

(A) The differentially expressed genes in adaptive versus parental HCC cells were determined by label-free fast quantitative analysis, and the mitochondrial-related candidates were further enriched using MitoCarta database 3.0. (https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways).

(B) Heatmap showing the abundance of proteins (LFQ intensity) identified in a label-free fast quantitative analysis. (|Fold change| > 1, Unique peptides >2, Score>20).

(C) Immunoblots analysis of TOMM34 expression in parental and metformin adaptive HCC cells.

(D) Relative growth rates of HCC cells at indicated times were assessed by MTT assay in the absence of metformin (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). (n=4 biological replicates, Two-way ANOVA).

(E) The relative number of living cells showing the effects of TOMM34 on the viability of HCC cells treated with the indicated concentration of metformin for 24 hours (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). (n=4 biological replicates, Two-way ANOVA).

(F) Trypan blue assay showing the survival of indicated cells treated with 20 mM metformin for 24 hours (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). (n=4 biological replicates, Two-way ANOVA).

(G) The morphology (Left), migration (Middle) and invasion (Right) of metformin adaptive Huh7 cells with (shTOM) or without (shNC) knockdown of TOMM34 are shown (5×104 cells for Transwell assay). Scale bars, (Left) 25 μm, (Middle) and (Right) 100 μm. (n=3 biological replicates, Two-way ANOVA).

(H) Wound healing assay showing the migration of metformin adaptive Huh7 cells with (shTOM) or without (shNC) TOMM34 knockdown. Scale bars, 200 μm. (n=4 technical replicates, Student's t-test).

(I) A tail vein injection model was established using adaptive Huh7 cells to assess cell migratory ability. Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=6 mice for each group, Student's t-test).

(J-K) The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=5 mice for each group, Student's t-test).

(L) Western blot analysis showing the effects of TOMM34 on the expression of EMT markers in HCC cells (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).

(M) The protein expression of TOMM34 in clinical HCC samples with or without metastasis. Scale bars, (Left) 100 μm, (Right) 25 μm. (n = 33 in metastasis present group, n = 53 in metastasis absent group, Student's t-test).

Data information: Data are presented as means ± S.D.

Source data are available online for this figure.
Disrupting metformin adaptation of liver cancer cells by targeting  the TOMM34/ATP5B axis (2022)
Figure 3

Fig. 3. TOMM34 preserves mitochondrial OXPHOS and ATP production in HCC cells.

(A-B) KEGG and GSEA (Gene Set Enrichment Analysis) analysis of the RNA-seq data identified the oxidative phosphorylation pathway as the one of most enriched signaling regulated by TOMM34. (P<0.020 in (A), P=0.0075 in (B)).

(C) Transmission electron microscopy (TEM) of Huh7 shNC and shTOM (shTOMM34) cells. Yellow arrowheads indicate mitochondria, bule arrowheads indicate nucleus, red arrowheads indicate endoplasmic reticulum. Scale bars, (Left) 20 μm, (Middle) 4 μm, (Right) 1 μm. (n=5 biological replicates, One-way ANOVA).

(D) mtDNA content in HCC cells (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). (n=4 biological replicates, Student's t-test).

(E) Intracellular ATP levels in HCC cells (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). (n=6 biological replicates in Huh7 cells, n=3 biological replicates in MHCC97H cells, n=5 biological replicates in PLC/PRF/5 cells, Student's t-test).

(F) Oxygen consumption rate (OCR) in Huh7 and HMCC97H cells with (shTOM) or without (shNC) TOMM34 knockdown was measured using a Seahorse XF24 Extracellular Analyzer (Oligo: oligomycin; FCCP: Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; R/A: rotenone/antimycin). (n=3 biological replicates).

(G) Western blot analysis showing the protein expression of the main mitochondrial respiratory chain complex subunit (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).

(H) Mitochondrial respiratory chain complex activity in HCC cells with or without TOMM34 knockdown/overexpression (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). (n=3 technical replicates, Two-way ANOVA).

(I) Intracellular ATP levels in adaptive Huh7 cells normalized to cells with (shTOM) or without (shNC) TOMM34 knockdown. (n=4 biological replicates, Two-way ANOVA).

(J) Detection of mitochondrial respiratory chain complex activity in metformin adaptive HCC cells with (shTOM) or without (shNC) TOMM34 knockdown. (n=4 technical replicates, Two-way ANOVA).

Data information: Data are presented as means ± S.D.

Source data are available online for this figure.
Disrupting metformin adaptation of liver cancer cells by targeting  the TOMM34/ATP5B axis (2022)
Figure 4

Fig. 4. TOMM34 interacts and positively correlates with ATP5B.

(A) Mass spectrometry and PrePPI dataset (https://bhapp.c2b2.columbia.edu/PrePPI/) identify the bait protein TOMM34 and its binding partner ATP5B.

(B) Co-IP assays indicate the interaction between exogenous TOMM34 and ATP5B in HEK293T cells.

(C) Co-IP assays indicate the interaction between endogenous TOMM34 and ATP5B in Huh7 HCC cells.

(D) The in situ interaction between TOMM34 and ATP5B was determined by proximity ligation assays (PLA). Scale bars, 10 μm.

(E) The mRNA and protein expression of ATP5B in indicated cells were determined by qPCR (n=3 technical replicates, Two-way ANOVA) or immunoblotting (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34-overexpressed cells).

(F-G) Western blot showing protein level of ATP5B in Huh7 (F) or PLC/PRF/5 (G) cells treated with or without 10 mg/mL CHX for 12 hours or 25 μM MG132 for 8 hours. Relative grey value of ATP5B was calculated using Image J (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells; CHX, cyclohexane). (n=3 technical replicates, Student's t-test).

(H) Tissue immunofluorescence assay showing the co-localization of TOMM34 and ATP5B at the invasive front of clinical HCC tissues. Scale bars, 20 μm.

(I) A positive correlation between TOMM34 and ATP5B levels in human HCC samples was shown by representative immunohistochemical images (Clinicopathologic characteristics are list in Appendix table S1). Scale bars, 50 μm. (n=72).

Data information: Data are presented as means ± S.D.

.
Disrupting metformin adaptation of liver cancer cells by targeting  the TOMM34/ATP5B axis (2022)
Figure 5

Fig. 5. ATP5B is essential for TOMM34-mediated metformin adaptation and HCC metastasis.

(A) ATP levels in adaptive HCC cells were measured with or without silencing of ATP5B by siRNAs. (n=4 biological replicates, One-way ANOVA)

(B-C) Western blot showing the expression of indicated proteins in adaptive HCC cells with or without ATP5B silencing.

(D-E) Transwell assays showing the migration and invasion of adaptive HCC cells with or without the silence of ATP5B (5×104 adaptive Huh7 cells, 1×105 adaptive PLC/PRF/5 cells), Scale bars, 100 μm. (n=3 biological replicates, Two-way ANOVA).

(F) ATP level of indicated cells was measured by ATP detection kit. (n=4 biological replicates, One-way ANOVA).

(G) Western blot showing the expression of EMT markers in cells with (shTOM) or without (shNC) knockdown of TOMM34 or overexpression of ATP5B.

(H) Transwell assays showing the migration and invasion of indicated cells (5×104 cells; shTOM, shTOMM34, TOMM34 knockdown cells). Scale bars, 100 μm. (n=4 biological replicates, two-way ANOVA).

Data information: Data are presented as means ± S.D.

Source data are available online for this figure.
Disrupting metformin adaptation of liver cancer cells by targeting  the TOMM34/ATP5B axis (2022)
Figure 6

Fig. 6. Gboxin abrogates metabolic adaptation to metformin in vitro and in vivo. (A-B) Co-IP assay was performed to detect the interaction between TOMM34 and ATP5B treated with or without Gboxin for 24 hours in parental or adaptive PLC/PRF/5 cells.

(C-D) Immunoblotting of EMT markers of HCC cells treated with or without 200 nM Gboxin for 24 hours (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).

(E-F) Transwell assay showing the migration and invasion of HCC cells treated with or without 200 nM Gboxin for 36 hours (5×104 cells; shTOM: shTOMM34, TOMM34 knockdown cells). Scale bars, 100 μm. (n=3 biological replicates, two-way ANOVA).

(G) Cellular ATP levels of HCC cells treated with or without 200 nM Gboxin for 24 hours (OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). (n=6 biological replicates in Huh7, n=4 biological replicates in PLC/PRF/5, one-way ANOVA).

(H) Adaptive Huh7 cells were subcutaneously injected into mice. The mice then received Gboxin, metformin or combinational treatment. The image and weight of tumor xenografts were shown. Scale bars, 1 cm. (n=6 mice in each group, one-way ANOVA).

(I) Images and weights of isolated tumors from HCC PDX models. Scale bars, 1 cm. (n=6 mice in each group, one-way ANOVA).

(J) Circulating tumor cell (CTC) was detected in HCC PDX models. (n=3 biological replicates, one-way ANOVA).

Data information: Data are presented as means ± S.D.

Source data are available online for this figure.

Expanded view figure legends
Coupling of ATPase activity, microtubule binding and mechanics in the dynein motor domain (2019)
Figure 1

Figure 1. Single-molecule motility properties of dynein stalk mutants reveal mutants with nucleotide-independent diffusive motility.

A. Structure and domain organization of the motor domain of cytoplasmic dynein (PDB 4RH7 (Schmidt et al, 2015)). Inset shows zoom of MTBD (white) and coiled-coil stalk (grey), which consist of two helices, CC1 and CC2. Well conserved residues, that were used as anchor points to define CC1 and CC2 in this study are depicted as red spheres. Numbering is based on yeast cytoplasmic dynein.

B. Table showing location, number of inserted or deleted residues and motility phenotype of all 18 stalk mutants. Examples of single-molecule assay results are shown in Figure EV2C and Appendix Figure S3. Sequence information and exact position of individual mutants are shown in Appendix Figure S1, S2. Quantification and classification are based on three technical repetitions.

C. Example kymographs for 'Directional - WT like', 'Diffusive like', 'No movement', and 'Transient binding'. Magnified area for 'Diffusive like' motion shows run of a single molecule. Kymographs for each mutant are shown in Figure EV2C.

D. Table showing the type of movement found for wild-type, mutant 2, mutant 5, and mutant 13 in a modified single-molecule assay with and without ATP. Classification of type of movement is based on two repetitions of different dynein preparations. Kymographs for each mutant are shown in Appendix Figure S4. Movies EV1-EV4 and EV5-EV8 show motility of wild-type and diffusive mutants with and without ATP, respectively.
Coupling of ATPase activity, microtubule binding and mechanics in the dynein motor domain (2019)
Figure 2

Figure 2. Diffusive mutants show microtubule-independent, high basal ATPase activity and low affinity for microtubules.

A-D Microtubule affinity measured by a cosedimentation assay in the apo state (full line) and in the presence of ATP (dashed line), and AMPPNP (dotted line) for wild-type (A), mutant 2 (B), mutant 5 (C), and mutant 13 (D). Appendix Table S2 shows fit equation and rate quantification for microtubule affinity data.

E. Microtubule stimulated ATPase activity of wild-type (grey), mutant 2 (red), mutant 5 (orange), and mutant 13 (purple). Appendix Table S4 shows fit equation and rate quantification for ATPase data.

F. Bar plot of basal ATPase activity of wild-type (grey), mutant 2 (red), mutant 5 (orange), and mutant 13 (purple).

Data information: Error bars show standard deviation of three repetitions of different dynein preparations.
HUWE1 interacts with PCNA to alleviate replication stress (2016)
Figure 1

Figure 1. HUWE1-knockout cells show genomic instability and increased replication stress. A, B. Western blot showing the absence of HUWE1 protein in 293T (A) and HeLa (B) cells subjected to CRISPR/Cas9-mediated HUWE1-deletion. C, D. HUWE1-knockout 293T (C) and HeLa (D) cells show increased DNA breaks in the absence of exogenous DNA damage treatment. Results from the Alkaline Comet Assay are shown. The “n” numbers of comet tails analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs. HUWE1-knockout HeLa cells did not show increases breakage in the Neutral Comet assay (Fig EV1B), indicating that the majority of breaks observed in cycling HUWE1-knockout cells are not double strand breaks, but rather single strand breaks and other types of lesions. E, F. Increased S-phase arrest in HUWE1-knockout 293T (E) and HeLa (F) cells. Cycling cells were incubated with BrdU and subjected to BrdU/PI bi-dimensional flow cytometry. Representative flow cytometry profiles are presented in Fig EV1D-F. Bars represent the fold increase in the percentage of cells with S-phase DNA content (between 2N and 4N) but negative for BrdU staining. Bars represent the average of three independent experiments, with error bars showing SD. The p-values are 0.0091 (E) and 0.0007 (F). G, H. The DNA fiber assay shows reduced replication tract length in HUWE1-knockout 293T (G) and HeLa (H) cells in the absence of exogenous DNA damage treatment. The “n” numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-values are 9.8x10-22 (G) and 1.0x10-10 (H).
HUWE1 interacts with PCNA to alleviate replication stress (2016)
Figure 2

Figure 2. Increased replication stress in HUWE1-knockdown cells. A. HUWE1 levels are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T cells. B. Quantification of HUWE1 expression following siRNA treatment. Band intensity was quantified using ImageJ software and normalized to Tubulin loading control. The average of three experiments is shown. Error bars indicate SD. C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N). D. Quantification of S-phase cells. Percentage of cells in R1 region is shown. Bars represent the average of three independent experiments. Error bars indicate SD. P-value is 0.0014. E. Quantification of S-phase arrested cells. Bars represent the fold increase in the percentage of cells in R2 region, normalized to siControl-treated cells. The average of three independent experiments is shown. Error bars indicate SD. P-value is 0.0135. F. DNA fiber experiment showing reduced replication tract length in HUWE1-depleted HeLa cells in the absence of exogenous DNA damage treatment. The “n” numbers of fibers analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs. G, H. Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV (G), and HU (H). As controls, both cells transfected with non-targeting siRNA (siControl), and non- transfected cells, were used. Note that the siHUWE1#2 oligonucleotide shows a stronger downregulation of HUWE1, and confers increased sensitivity compared to siHUWE1#1. The average of nine experiments is shown. Error bars represent SEM. P-values (representing the statistical difference between the samples at the highest dose treatment) are: 4.55x10-9 (G) and 1.8x10-8 (H). I. DNA fiber experiments showing decreased replication tract length following UV-B treatment (30J/m2) in HUWE1-depleted HeLa cells. The “n” numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-value is 2.11x10-20.
HUWE1 interacts with PCNA to alleviate replication stress (2016)
Figure 3

Figure 3. HUWE1 localizes to replication forks. A. iPOND experiment showing that HUWE1 localizes to replication forks in 293T cells. Binding to nascent DNA was increased after UV exposure. Because of the difficulties in removing all the crosslinks in high molecular weight proteins, HUWE1 is detected as a high molecular weight smear. A control experiment using the HUWE1-knockout cells (shown in the right side panel) confirmed the specificity of the HUWE1 signal. B. Immunofluorescence experiment showing that HUWE1 localizes to chromatin foci in HeLa cells exposed to replication fork stalling agents HU (2mM for 16h) and UV (40J/m2, analyzed 2h later). C. Co-immunofluorescence experiments showing co-localization of HUWE1 and PCNA in chromatin foci. U2OS cells were analyzed 2h after exposure to UV (40J/m2). Quantifications of co-localization, and additional micrographs are presented in Fig EV2A-D.
HUWE1 interacts with PCNA to alleviate replication stress (2016)
Figure 4

Figure 4. HUWE1 interacts with PCNA. A. Schematic representation of full-length HUWE1. Shown is the PIP-box domain we describe here (aa3880-3887). ARLD: Armadillo repeat-like domain; UBA: Ubiquitin-associated domain; BH3: Bcl2-homology 3 domain; HECT: Homologous to the E6-AP Carboxyl Terminus (catalytic ubiquitin ligase domain). The span of the HUWE1C-ter (3875-end) fragment used in subsequent studies is also indicated. B-D. Interaction between endogenous HUWE1 and PCNA in 293T cells. B. Anti-PCNA immunoprecipitation, showing that HUWE1 co-precipitates. C. Reciprocal experiment showing that PCNA co-precipitates with HUWE1 in anti-HUWE1 immunoprecipitation. D. As control, endogenous HUWE1 was depleted by siRNA treatment, and extracts were subjected to anti-HUWE1 immunoprecipitation. The HUWE1 blot shows that much less HUWE1 is precipitated by anti-HUWE1 antibodies from HUWE1-knockdown cells than control, as expected. PCNA is co-precipitated by anti-HUWE1 antibodies from control, but not HUWE1-depleted cells, showing that the interaction is specific and not due to unspecific binding of PCNA to anti-HUWE1 antibodies. E, F. HUWE1 co-precipitates with PCNA in anti-PCNA immunoprecipitation from extracts of MCF7 (E) and 8988T (F) cells, showing that the HUWE1-PCNA interaction can be detected in different cell lines. G. Co-immunoprecipitation experiment from control and RAD18-knockout 293T cells, showing that the HUWE1-PCNA interaction is not affected by loss of PCNA ubiquitination. RAD18-knockout cells were obtained by CRISPR/Cas9 technology. The RAD18 and PCNA blots show loss of RAD18, and of PCNA ubiquitination respectively. H. Myc-HUWE1C-ter fragment was transfected in 293T cells. Anti-PCNA immunoprecipitation showed that the C-terminal HUWE1 fragment interacts with PCNA. I. Coomassie staining of recombinant GST-PCNA expressed and purified from bacteria. J. GST-pulldowns using GST-PCNA or GST-empty as control, and extracts of 293T cells transfected with Myc-HUWE1C-ter, showing interaction of this fragment with recombinant PCNA.
HUWE1 interacts with PCNA to alleviate replication stress (2016)
Figure 5

Figure 5. The PIP-box of HUWE1 is essential for its interaction with PCNA. A, B. GST-pulldowns showing that HUWE1 PIP-box mutants do not interact with PCNA. A. Recombinant GST-tagged HUWE1C-ter, either wild-type or PIP-box mutant variants (QVFF and VFF –the indicated residues, critical for the PIP-box, were mutated to A), were purified from bacteria (see Coomassie staining) and employed for GST-pulldowns using extracts of 293T cells. PCNA co-precipitated with wild-type, but not PIP-box mutants. B. Reciprocal GST-pulldown using recombinant GST-PCNA and extracts of 293T cells overexpressing Myc-HUWE1C-ter. Wild-type, but not the PIP-box mutant (VFF) HUWE1 fragment bound to GST-PCNA. C, D. Reciprocal co-immunoprecipitation experiments showing that PIP-box mutation abolishes PCNA interaction. C. 293T cells were transfected with Myc-HUWE1C-ter variants, and subjected to anti-PCNA (C) or anti-Myc (D) immunoprecipitation. Only wild-type, but not PIP-box mutant (VFF) HUWE1 interacted with PCNA in both experimental setups. E. Mutation of the PIP-box in full-length HUWE1 also blocks PCNA interaction. Full-length Myc-tagged HUWE1 (wild-type or PIP-mutant) was transfected in 293T cells. Following Myc-immunoprecipitation, endogenous PCNA was detected in wild-type, but not PIP-box mutant (FF) complexes.
HUWE1 interacts with PCNA to alleviate replication stress (2016)
Figure 6

Figure 6. HUWE1 interaction with PCNA is essential for alleviating replication stress. A. Stable expression of Myc-tagged wild-type, PIP-box mutant, and catalytic inactive HUWE1 variants in HUWE1-knockout 293T cells. The Western blot shows that expression of the Myc-tagged variants stably transfected in the knockout cell lines, is similar to the endogenous HUWE1 expression. B. Myc-tag immunoprecipitation using extracts of the corrected cell lines described above. As expected, PCNA co-precipitates with wild-type, but not PIP-box mutant HUWE1 (FF). C. iPOND experiment using HUWE1-knockout 293T cells, corrected with wild-type or PIP-mutant HUWE1. The PIP-mutant shows reduced crosslinking to nascent DNA, indicated defective recruitment to replication forks. D. Clonogenic assay showing that stable expression of wild-type HUWE1, but not of the PIP box mutant or the catalytic mutant, corrects the HU sensitivity of the HUWE1-knockout 293T cells. Shown is the average of 3 independent experiments -/+SD. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T (for the 0.5mM HU condition). P-values are: 0.0003 (293T vs HUWE1-/-; 0.157 (293T vs HUWE1-/- +WT); 0.0045 (293T vs HUWE1-/- +FF); 0.0006 (293T vs HUWE1-/- +C4341A; 0.047 (HUWE1-/- +WT vs HUWE1-/- +FF). E. The S-phase arrest phenotype is also rescued by wild-type, but not PIP-mutant or catalytic mutant HUWE1. S-phase arrested cells were quantified using the BrdU/PI bi-dimensional flow cytometry assay. Shown is the average of 3 independent experiments -/+SD. The p-values shown specify the statistical significance relative to 293T (0.018, 0.957, 0.015, and 0.024 respectively). A quantification of the same data, showing the percentage of cells arrested in S-phase, is shown in Fig EV3A. F. Normal replication tract length, quantified using the DNA fiber assay, is restored by expressing wild-type, but not PIP-mutant HUWE1, in the HUWE1-knockout 293T cells. The “n” numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T. P-values are: 1.47x10-11 (293T vs HUWE-/- samples), 0.094 (293T vs HUWE-/- +WT samples), 1.40x10-7 (293T vs HUWE-/- +FF samples), 1.22x10-7 (HUWE-/- vs HUWE-/- +WT samples), 0.679 (HUWE-/- vs HUWE-/- +FF samples). G. Alkaline Comet Assay showing that wild-type, but not the FF mutant can correct the breakage phenotype of HUWE1-knockout 293T cells. The “n” numbers of comet tails analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. The p-values indicated are: 4.18x10-43 (293T vs HUWE-/- samples), 6.89x10-41 (HUWE1-/- +WT vs FF samples), and 5.97x10-50 (HUWE1-/- +WT vs C4341A samples).