10 mg/mL X-gal substrate in DMF, 2.5 µL of staining supplement and 235 µL of staining solution) per well, overnight at 37°C. PBS was added to the empty wells in the plate to avoid the evaporation of the staining solution mix. Cells were observed under the microscope for development of blue color, and the reaction was stopped by removal of the staining solution mix and rinsed with PBS three times. Coverslips were mounted in mounting medium (S3023, Agilent) and slides were observed with a Leica DM750 upright brightfield microscope equipped with a Leica ICC50W digital color camera. A minimum of five areas per coverslip were considered so that more than 200 cells per coverslip were manually counted using an ×10 magnification objective. The number of SA β-gal positive cells was normalized by the total number of cells.

## Tom20 immunofluorescent staining

Mitochondrial network was assessed by Tom20 immunolabeling.<sup>158</sup> Cells were seeded over 12-mm coverslips (631-1577P, VWR) in tissue-culture treated 24-well plates and allowed to grow until they reached 80% confluency. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min and washed with PBS. Blocking was performed in 1% BSA and 2% FBS-PBS during 30 min. Next, cells were incubated with Tom20 antibody (sc-11415, Santa Cruz Biotechnology) diluted 1:200 in blocking solution for 1 h, followed by washes with PBS and incubation with Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-21206, Invitrogen) diluted 1:500 in blocking buffer during 30 min. DNA and actin F cytoskeleton were stained with DAPI (D1306, Invitrogen) and Rhodamine-Phalloidin (R415, Invitrogen), respectively. Finally, coverslips were rinsed three times with PBS and mounted using ProLong Gold Antifade Mountant (P10144, Molecular Probes). Images were acquired in a Nikon Ti Eclipse confocal microscope (Nikon Instruments). Hardware and image acquisition were controlled by NIS-Elements imaging software (Nikon Instruments). The pipeline analysis of mitochondrial morphology and mass was adapted in Fiji software from Koopman et al.<sup>91</sup>

## Transmission electron microscopy (TEM) of epon-embedded ultrathin sections

Cells were seeded in 10 cm tissue-culture treated dishes and allowed to grow until they reached 80-100% confluency. Cells were fixed with equal parts of a 4% glutaraldehyde solution (49625, Sigma-Aldrich) in 0.24 M PBS pH 7.2 and cell culture medium during 2 h at RT. Cells were gently collected with a scraper, transferred to 15 mL tubes and centrifuged (1,000 x g, 5 min). The fixative solution was discarded and the pellet was resuspended in 1 mL of 0.12 M PBS pH 7.2 and transferred to a new tube. A compact pellet was generated by centrifugation at 5,000 x g 5 min and embedded in epoxy resins. After polymerization, 150 nm thick sections were obtained using an ultramicrotome (Leica Microsystems) and a diamond knife (Diatome), placed on 100 mesh hexagonal Cu/Pd EM grids, stained with uranyl acetate and counterstained with lead citrate to reveal and enhance contrast of cellular membranes. For morphological analysis, ultrathin epon-embedded cell sections were studied by TEM. Images were collected using a JEOL JEM-1230 transmission electron microscope operating at 100 kV and equipped with an Ultrascan 4000S P 4 K × 4 K CCD camera (GATAN). Images were acquired at different magnifications ranging from 1,000× to 20,000X. Analysis and segmentation of mitochondria and ER were performed using images with 2,500X and 5,000× magnification. A total of 56 micrographs per condition were analyzed. Images were first subjected to contrast-limited adaptive histogram equalization (CLAHE) to ensure homogeneity in black and white balance through all micrographs using the Microscopy Image Browser (MIB) standalone version 2.7 software.<sup>159</sup> Then segmentation masks were manually applied on the cell sites where mitochondria and ER were identified in addition to using local thresholding. Total cell area, total mitochondria area, total ER area, mitochondria major and minor axis, and number of mitochondria per visualized cell were estimated within the region of interest.

## MitoTracker Green staining

Cells were seeded in tissue-culture treated 6-well plates and allowed to grow until they reached 80% confluency. Mitochondrial mass was assessed by fluorescent labeling with MitoTracker Green FM probe (M7514, Invitrogen) in combination with LIVE/DEAD fixable blue dead cell stain kit (L23105, Invitrogen), by following the manufacturer's recommendations. Culture medium was removed, cells were washed with PBS twice, detached by trypsinization and transferred to 15 mL tubes. Each cell pellet was washed with 5 mL of PBS and collected by centrifugation (600 x g, 5 min). Next, each pellet was resuspended in 100 µL of the LIVE/DEAD cell dye in PBS and incubated for 10 min in the dark at RT. The staining solution was diluted by adding 500 µL of PBS per tube and cells were collected by centrifugation (600 x g, 5 min). Each pellet was resuspended in 100 µL of 100 nM MitoTracker Green FM probe in PBS and incubated 45 min at RT protected from the light. The staining solution was diluted by adding 500 µL of PBS per tube and cells were collected by centrifugation (600 x g, 5 min). Each cell pellet was resuspended in 200 µL of ice-cold 4% paraformaldehyde solution in PBS (sc-281692, Santa Cruz Biotechnology) and fixed during 15 min in the dark at 4°C. Fixation was stopped by the addition of 1 mL of PBS supplemented with 5% FBS per tube, and cells were collected by centrifugation (600 x g, 5 min, 4°C). Each cell pellet was finally resuspended in 200 µL of 5% FBS-PBS. MitoTracker Green FM probe fluorescence (λ<sub>ex</sub> = 490 nm, λ<sub>em</sub> = 516 nm) was assayed by flow cytometry in a BD FACSympathy system (BD Biosciences). A set of unstained cells were used as a blank. The results were analyzed with FlowJo v10 software (BD Biosciences) and the geometric mean fluorescence intensity (gMFI) was calculated.

## Mitochondrial energetic metabolism studies

A two-step seeding process was followed to ensure that cells were evenly distributed throughout the wells of Seahorse XF24 cell culture microplates (102070-001, Agilent). First, 100 µL of the cell suspension were seeded per well and the plates were allowed
to rest in the hood for 1 h at RT. In a second step, each well was topped up with 150 µL of culture medium and cells were allowed to grow until they reached 80% confluency. Mitochondrial respiration was assessed by performing a Cell Mito Stress Test in a Seahorse XF24 Analyzer (Agilent). The day before the assay, the XF24 sensor cartridges (102070-001, Agilent) were hydrated with Seahorse XF calibrant solution (102070-001, Agilent) overnight at 37°C in a 0% CO₂ incubator. On the day of the assay, culture medium was replaced with bicarbonate-free low-buffered assay medium and cells were incubated for 1 h at 37°C in a 0% CO₂ atmosphere. After establishing a respiration baseline, 6 µM oligomycin, 3 µM carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP) and a combination of 0.5 µM antimycin A and 0.5 µM rotenone were sequentially injected through the reagent ports of the cartridges in order to measure changes in the oxygen consumption rate (OCR) related to ATP-linked, maximum and non-mitochondrial respiration, respectively. The OCR, expressed as pmol of O₂/min, was normalized by the number of cells or protein content, which were estimated by crystal violet or Micro BCA Protein Assay Kit, respectively.

## MitoSOX red staining

Cells were seeded over 12-mm coverslips (631-1577P, VWR) previously coated with a 0.01% poly-L-lysine solution (P4707, Sigma-Aldrich), in tissue-culture treated 24-well plates and allowed to grow until they reached 80% confluency. Mitochondrial reactive oxygen species (ROS) production was measured by MitoSOX Red mitochondrial superoxide indicator (M36008, Invitrogen), according to the manufacturer's instructions. MitoSOX Red is a fluorogenic dye which is selectively targeted to mitochondria and exhibits red fluorescence when oxidized by superoxide anion. Cells were washed twice with warm DPBS, calcium, magnesium (14040133, Invitrogen) and incubated with 500 µL of 1 µM MitoSOX Red reagent diluted in DPBS, calcium, magnesium for 10 min at 37°C protected from the light. An unstained well was used as blank. Cells were washed with DPBS, calcium, magnesium three times and fixed in ice-cold 4% paraformaldehyde solution in PBS (sc-281692, Santa Cruz Biotechnology) for 10 min at RT. The fixative solution was removed after two washes with DPBS, calcium, magnesium and coverslips were mounted in Fluoroshield with DAPI mounting medium (F6057, Sigma-Aldrich). MitoSOX Red fluorescence (λ<sub>ex</sub> = 510 nm, λ<sub>em</sub> = 580 nm) was observed in the Axio Imager D1 epifluorescent microscope (Zeiss). A minimum of five areas per coverslip were assessed using an ×40 objective. MitoSOX Red fluorescence intensity was quantified using Fiji software (https://imagej.net/software/fiji/) and normalized by the number of nuclei stained with DAPI.

## Tetramethylrhodamine (TMRE) staining

Cells were seeded in tissue-culture treated 24-well plates and allowed to grow until they reached 80% confluency. Mitochondrial membrane potential was estimated by Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (T669, Invitrogen), by following the manufacturer's indications. TMRE is a fluorogenic cationic dye which is selectively targeted to active mitochondria. Cells were washed twice with warm DPBS, calcium, magnesium (14040133, Invitrogen) and incubated with 500 µL of 0.5 µM TMRE probe diluted in DPBS, calcium, magnesium for 30 min at 37°C protected from the light. An unstained well was used as blank. Cells were washed with DPBS, calcium, magnesium twice and maintained in 300 µL DPBS, calcium, magnesium. TMRE fluorescence (λ<sub>ex</sub> = 548 nm, λ<sub>em</sub> = 574 nm) was measured in a SpectraMax M2/M2e microplate reader (Molecular Devices). TMRE probe fluorescence was normalized with the total protein content of each well, which was determined by the Micro BCA Protein Assay Kit.

## ATP levels quantification

Cells were seeded in tissue-culture treated 12-well plates and allowed to grow until they reached 80% confluency. Cellular ATP levels were determined with the ATPlite luminescence assay system (6016943, PerkinElmer), by following the manufacturer's instructions. The assay was performed in OptiPlate-96 white opaque 96-well microplates (6005290, PerkinElmer) and luminescence was measured in a Veritas microplate luminometer (Turner BioSystems). ATP concentration was calculated by interpolation to an ATP standard curve and subsequent normalization with the total protein content of each sample, which was determined by the Bradford assay.

## NAD+/NADH measurement

Cells were seeded in tissue-culture treated 6-well plates and allowed to grow until they reached 80% confluency. Cellular NAD+/NADH levels were determined with the NAD/NADH Colorimetric Assay Kit (ab65348, Abcam), according to the manufacturer's instructions. The assay was performed in 96-well clear flat bottom plates and absorbance at 450 nm was measured in a SpectraMax M2/M2e microplate reader (Molecular Devices) after 2 h incubation at RT with NAD cycling enzyme mix. Total NAD and NADH concentrations were calculated by interpolation to a NADH standard curve. The levels of NAD+ were calculated by subtracting NADH from total NAD, and the NAD+/NADH were represented.

## Cytosolic calcium (Ca²⁺) concentration determination

Cells were seeded over 12-mm coverslips (631-1577P, VWR) previously coated with a 0.01% poly-L-lysine solution (P4707, Sigma-Aldrich), in tissue-culture treated 24-well plates and allowed to grow until they reached 80% confluency. Cytosolic Ca²⁺ levels were determined by using Fura-2-AM (F1201, Invitrogen).¹⁶⁰,¹⁶¹ Fura-2 is a cell-permeable ratiometric cytosolic Ca²⁺ indicator, whose excitation wavelength shifts from 380 nm to 340 nm as it binds to Ca²⁺, while its emission maximum is independent of Ca²⁺ concentration. Cells were washed twice with 0% FBS-culture medium and incubated with 1 µM Fura-2 dissolved in 0% FBS-culture medium
for 30–45 min at 37°C. After loading, cells were incubated in 0% FBS-culture medium for 15 min at 37°C protected from the light. Coverslips were washed with 20 mM Tris-HCl pH 7.4, 2.4 mM CaCl₂, 10 mM glucose solution and mounted on a thermostatized mi-cro-perfusion chamber. Single-cell Fura-2 excitation intensity ratio (λ<sub>ex</sub> = 340 and 380 nm, λ<sub>em</sub> = 510 nm) was measured with an ×40 oil-immersion magnification objective in an Eclipse TE 300-based microspectrofluorometer (Nikon) coupled to a DeltaRAM illumina-tion system (Photon Technologies International). After recording a baseline for 30 s, 10 µM thapsigargin (T9033, Sigma-Aldrich) or 100 nM ATP was added to the medium to trigger the release of Ca²⁺ from the endoplasmic reticulum (ER) for 2 min and finally Ca²⁺ was added to measure the entrance of extracellular Ca²⁺ for additional 2 min. The ratio of excitation intensities at 340 nm and 380 nm (R) was related to Ca²⁺ levels by the following equation: [Ca²⁺]= K<sub>d</sub> x Q x (R - R<sub>min</sub>)/(R<sub>max</sub> - R). R<sub>min</sub> and R<sub>max</sub> refer to the fluorescence intensity ratios when the probe is free or completely saturated of Ca²⁺ respectively, Q represents the ratio of min-imum to maximum fluorescence at 380 nm (F<sub>min</sub>/F<sub>max</sub>), and K<sub>d</sub> is the Ca²⁺ dissociation constant of Fura-2. The values for F<sub>min</sub>, F<sub>max</sub>, R<sub>min</sub>, R<sub>max</sub> and K<sub>d</sub> were previously determined by means of a Fura-2 calibration curve in the presence of known Ca²⁺ concentrations.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed using Prism 8 software (GraphPad). Statistical details of experiments can be found in the figures and corresponding legends. Unless otherwise stated, experiments were reproduced at least three times. Data are represented as the mean ± standard deviation (SD) of at least three biological replicates within one representative experiment. A two-tailed t test was used to compare the differences between two groups. A p value <0.05 was considered statistically significant for all analyses and defined as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. If not indicated otherwise, the differences were not significant (n.s.).

ADDITIONAL RESOURCES

Animations

Animations were created using BioRender software (https://biorender.com).
Supplemental information

SUMOylation controls Hu antigen R

posttranscriptional activity in liver cancer

Sofia Lachiondo-Ortega, Claudia M. Rejano-Gordillo, Jorge Simon, Fernando Lopitz-Otsoa, Teresa C. Delgado, Krystyna Mazan-Mamczarz, Naroa Goikoetxea-Usandizaga, L. Estefanía Zapata-Pavas, Ana García-del Río, Pietro Guerra, Patricia Peña-Sanfélix, Natalia Hermán-Sánchez, Ruba Al-Abdulla, Carmen Fernandez-Rodríguez, Mikel Azkargorta, Alejandro Velázquez-Cruz, Joris Guyon, César Martín, Juan Diego Zalamea, Leire Egia-Mendikute, Arantza Sanz-Parra, Marina Serrano-Maciá, Irene González-Recio, Monika Gonzalez-Lopez, Luis Alfonso Martínez-Cruz, Patrizia Pontisso, Ana M. Aransay, Rosa Barrio, James D. Sutherland, Nicola G.A. Abrescia, Félix Elortza, Amaia Lujambio, Jesus M. Banales, Raúl M. Luque, Manuel D. Gahete, Asís Palazón, Matías A. Avila, Jose J. G. Marin, Supriyo De, Thomas Daubon, Antonio Díaz-Quintana, Irene Díaz-Moreno, Myriam Gorospe, Manuel S. Rodríguez, and María Luz Martínez-Chantar
<figure><img src="image_71.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_73.png" /></figure>
**Figure S1. (A)** Detection of SUMOylated HuR in the tumor (T) and surrounding tissue (ST) of a cohort of HCC patients (n=5) after protein pulldown with GST control and western blotting analysis. ELAVL1/HuR and the main components of the SUMO pathway (B) mRNA and (C) protein expression levels and quantification of in the PLC/PRF/5 and HuH-7 human hepatoma cell lines.

(C) Data are represented as the mean ± SD of at least three biological replicates within one representative experiment. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 and \*\*\*\*p < 0.0001, two-tailed t-test. If not indicated otherwise, the differences were n.s. Western blots are representative of at least three biological replicates.

Related to **Figure 1**.
<figure><img src="image_74.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_75.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_76.png" /></figure>

**Figure S2.** H<sub>6</sub>-tagged SUMO and FLAG/RGS-tagged PIAS and SENPs protein expression levels after transient transfection of plasmids expressing (A) SUMO, (B) PIAS and (C) SENP isoforms, in addition to WT HuR and UBC9, in the mouse liver progenitor MLP-29 cell line.

(A-C) Western blots are representative of at least three biological replicates.

Related to **Figure 2**.
<figure><img src="image_77.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_82.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_83.png" /></figure>
**Figure S3.** (A) *Pias2b*, *Senp 1*, *2* and *3* mRNA expression levels after siRNA transfection in the MLP-29 cell line. V5-tagged HuR protein expression levels and its smear after co-transfection of plasmids expressing WT HuR, UBC9 and SUMO2/3 in addition to siRNAs silencing a subset of (B) *Pias* and (C) *Senp* isoforms in the MLP-29 cell line. (D) Modified V5-HuR protein enrichment after transient transfection of plasmids inducing the expression of WT HuR, UBC9 and SUMO2/3 in addition to siRNAs downregulating *Senp1* or *Senp2* expression and subsequent nickel-histidine affinity purification, relative to total V5-HuR protein expression levels in the MLP-29 cell line.

(A) Data are represented as the mean ± SD of at least three biological replicates within one representative experiment. \*p < 0.05 and \*\*p < 0.01, two-tailed t-test.

(B-D) Western blots are representative of at least three biological replicates.
Related to **Figure 2**.
<figure><img src="image_84.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_85.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_86.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_87.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_88.png" /></figure>

**Figure S4. (A)** Modified V5-HuR protein enrichment after transient transfection of plasmids expressing the different SUMO paralogs and subsequent nickel-histidine affinity purification, relative to total V5-HuR protein expression levels in the HuH-7
human hepatoma cell line. HuR protein expression levels and its smear after co-
transfection of plasmids expressing SUMO2/3 as well as the (B) PIAS and (C) SENP
isoforms in the HuH-7 cell line. H6-tagged SUMO2/3 and UBC9 protein expression
levels after transient transfection of plasmids expressing UBC9, SUMO2/3 and WT
HuR or a subset of lysine-to-arginine HuR mutants contained in the RRM1-2 domains
in the (D) MLP-29 and (E) HuH-7 cell lines.

(A-E) Western blots are representative of at least three biological replicates.

(D and E) The entire blot image was digitally processed to eliminate irrelevant lanes.

Related to **Figure 2**.
A

IP: α-V5

<figure><img src="image_89.png" /></figure>

B

<figure><img src="image_90.png" /></figure>

C

<figure><img src="image_91.png" /></figure>

D

<figure><img src="image_92.png" /></figure>

E

<figure><img src="image_93.png" /></figure>
**Figure S5.** (A) SUMO2/3-modified V5-HuR enrichment after transient transfection of plasmids expressing WT and K120/182R HuR under normoxic and hypoxic conditions and subsequent protein immunoprecipitation using anti-V5 antibody, relative to total V5-HuR protein expression levels in the HuH-7 human hepatoma cell line. (B) Ubiquitinated V5-HuR protein enrichment after transient transfection of plasmids inducing the expression of WT HuR and the different SUMOylation mutants as well as H<sub>6</sub>-tagged ubiquitin and downstream nickel-histidine affinity purification, relative to total V5-HuR protein expression levels in the HuH-7 cell line. (C) SUMO2/3 and HuR protein expression levels and quantification after incubating the HuH-7 cell line with 100 nM ML-792 SAE inhibitor for 4 h. (D) SUMO2/3-modified and (E) ubiquitinated V5-HuR enrichment after transient transfection of plasmids expressing HuR and H<sub>6</sub>-tagged SUMO2/3 or ubiquitin, respectively, in addition to treatment with 100 nM ML-792 for 4 h and subsequent nickel-histidine affinity purification, relative to total V5-HuR protein expression levels in the HuH-7 human hepatoma cell line.

(A-E) Western blots are representative of at least three biological replicates. (E) The entire blot image was digitally processed to eliminate irrelevant lanes.

(C) Data are represented as the mean ± SD of at least three biological replicates within one representative experiment. *p* < 0.05, two-tailed t-test.

Related to **Figure 2**.
<figure><img src="image_94.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_95.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /></figure>

<figure><img src="image_98.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_99.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_100.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_101.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_102.png" /></figure>
**Figure S6.** (A) Statistics of molecular dynamics (MD) computations. *Upper left panel*, time course for the RMSD values along the trajectories. *Black* stands for the untouched RRM1-2, *blue* for the construct SUMOylated at K182, *red* for SUMOylation at K120 and *green* for the double modification. *Lower left*, evolution of the radius of gyration of HuR RRM1-2 along the trajectories, excluding SUMO molecules. The colour code used is the same as in the *upper panel*. *Right panels* show the secondary structure of the HuR RRM12 construct, excluding SUMO molecules, along the trajectories computed for the unmodified (*upper*) and the doubly SUMOylated protein (*lower*). Colours indicate, according to the right legend, different secondary structures as defined in the DSSP program. (B) Contribution of key target residues to the total solvent accessible surface of the RRM1-2 moiety. Frequency distributions of accessible surfaces along the whole MD trajectories. All selected residues establish different kind of interactions with a poly-U RNA oligo, according to the XRD data.¹ (C) MD computations on the HuR RRM1-2 tandem with an 11-mer *c-fos* mRNA segment. Starting structure was that reported by Wang et al. (PDB code 4ED5).¹ Data in *black*, *blue* and *red* correspond to WT, K120R and K182R trajectories, respectively. The root-mean-square deviation (RMSD) values below 3 Å indicate that none of the domains undergoes any significant change, and that the two RRM domains keep their relative orientation. The radius of gyration was measured for the coordinates of both, protein and RNA (1,2, RNA), or only the protein moiety (1,2) along the whole trajectories. Radius of gyration (R<sub>G</sub>) values are lower when considering both, protein and RNA, since the latter is inserted in a cleft between the two domains and the linker, instead of covering an open surface. The constant R<sub>G</sub> values clearly indicate that the insertion of RNA between the two domains makes the whole complex rigid. The lack of differences between the WT construct and the mutant species is consistent with the fact that K120 locates far from the binding site, and that K182 only contributes with a single H-bond at the 5' end. (D) Recombinant HuR protein constructs expression and purity assessment by SDS-PAGE analysis. Coomassie Blue-stained 15% gel loaded with 25 µg of protein samples. The lanes show single bands slightly below 25 kDa, which is consistent with the theoretical molecular weight of the constructs (ca. 22.8 kDa). (E) Far-UV circular dichroism (CD) spectra of HuR RRM1-2 species at 10 µM. The WT protein profile is shown as a dashed line in all spectra, whereas the profile of the mutants is presented as a solid line superimposed on the WT spectrum for
comparison purposes. The CD measurements do not show significant differences
between HuR RRM1-2 constructs, suggesting that the secondary structure of all of
them is essentially identical. (F) Dynamic light scattering (DLS) analysis to study
protein homogeneity. Volume-weighted particle size distributions (PSDs) are given for
each HuR RRM1-2 construct at 1 mg/ml. The WT protein profile is shown as a dashed
line in all spectra, whereas the profile of the mutants is presented as a solid line
superimposed on the WT graph for comparison purposes. The graphs show nearly
identical PSDs for all constructs, demonstrating that the point mutations introduced
into HuR RRM1-2 have no effect on its aggregation state. (G) Isothermal titration
calorimetry (ITC) measurements of T-rich DNA binding to HuR RRM1-2 species. 150-
180 µM of each HuR RRM1-2 construct were titrated to 10-15 µM of T-rich DNA.
Thermograms and binding isotherms are shown in the *upper* and *lower* panels,
respectively. The curve through the points represents the best fit to a one-site binding
model. ITC data analysis provide very similar *K_D* values for all HuR RRM1-2
constructs, indicating that the SUMOylation mutants assayed preserve their ability to
bind nucleic acid virtually intact. (H) Derivation of binding and dissociation kinetics
from the Brownian dynamics (BD) rigid-body diffusion trajectories. For each set of
computations, two curves are represented, corresponding to two independent pair-
distances criteria for reaction.² *Upper graph*, binding rates for the WT (*green*) and
K120/182R (*red*). *Lower graph*, residence times at different distance cut-offs for the
two interactions. Error bars indicate standard deviation.

(D) Western blots are representative of at least three biological replicates.

Related to **Figure 2**.
<figure><img src="image_103.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_104.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_105.png" /></figure>

<figure><img src="image_106.png" /></figure>

**Figure S7. (A) V5-HuR and (B) total HuR protein expression levels and quantification after stable transfection of WT HuR as well as the K120/182R, K120R, K182R HuR mutant species in the HuH-7 cell line, relative to β-actin.**

(A and B) Data are represented as the mean ± SD of at least three biological replicates within one representative experiment. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 and \*\*\*\*p < 0.0001, two-tailed t-test vs. HuH-7 WT HuR. If not indicated otherwise, the differences were n.s.

Related to Figure 3.
A

<figure><img src="image_107.png" /></figure>

<table><thead><tr><th>p-value<br>Compared to HuH-7 WT HuR</th><th>24h</th><th>48h</th><th>72h</th><th>96h</th></tr></thead><tbody><tr><td>HuH-7 EV</td><td>n.s.</td><td>n.s.</td><td>n.s.</td><td>n.s.</td></tr><tr><td>HuH-7 HuR [K120/182R]</td><td>****</td><td>****</td><td>****</td><td>****</td></tr><tr><td>HuH-7 HuR [K120R]</td><td>****</td><td>****</td><td>****</td><td>****</td></tr><tr><td>HuH-7 HuR [K182R]</td><td>****</td><td>****</td><td>****</td><td>****</td></tr></tbody></table>

B

<figure><img src="image_108.png" /></figure>

<table><thead><tr><th>p-value<br>Compared to HuH-7 WT HuR</th><th>6h</th><th>12h</th><th>24h</th><th>36h</th><th>48h</th><th>60h</th></tr></thead><tbody><tr><td>HuH-7 EV</td><td>n.s.</td><td>n.s.</td><td>n.s.</td><td>n.s.</td><td>**</td><td>****</td></tr><tr><td>HuH-7 HuR [K120/182R]</td><td>***</td><td>****</td><td>****</td><td>****</td><td>****</td><td>****</td></tr><tr><td>HuH-7 HuR [K120R]</td><td>****</td><td>****</td><td>****</td><td>****</td><td>****</td><td>****</td></tr><tr><td>HuH-7 HuR [K182R]</td><td>*</td><td>***</td><td>****</td><td>****</td><td>****</td><td>****</td></tr></tbody></table>
**Figure S8.** Representative pictures and statistical significance of (A) cell proliferation and (B) scratch-wound process of HuH-7 cell lines stably expressing WT HuR and the different SUMOylation mutants analysed in the IncuCyte system.

(A-B) Images are representative of at least three biological replicates. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 and \*\*\*\*p < 0.0001, two-tailed t-test vs. HuH-7 WT HuR.

Related to **Figure 3**.
<figure><img src="image_109.png" /><figcaption>**Figure S9.** (A) Crystal violet staining of the HuH-7 cell lines stably expressing WT HuR and the K120/182R, K120R, K182R SUMOylation mutants treated with the indicated doses of palbociclib for 3 days. (B) Quantification of cell apoptosis in the</figcaption></figure>
HuH-7 cell lines stably expressing WT HuR and the different SUMOylation mutants after an acute 3-day treatment with a range of palbociclib concentrations, analysed by caspase 3 activity assay. (C) Representative pictures of senescence associated β-galactosidase staining in the HuH-7 cell lines stably expressing the WT and the K120/182R, K120R, K182R HuR mutant species after a chronic 2-week treatment with a range of palbociclib concentrations. (D) p-RB Ser780, RB, CCND1, CCNA2 and p-H2AX Ser139 protein expression levels and quantification in the HuH-7 cell lines stably expressing WT HuR and the K120/182R SUMOylation mutant after a chronic 2-week treatment with 5 nM palbociclib, relative to β-actin. In the heatmap, red indicates high protein expression levels, blue indicates low, and values outside the defined range are represented in black.

(A-C) Images are representative of at least three biological replicates. (C) Scale bar is 200 µm.

(B and D) Data are represented as the mean ± SD of at least three biological replicates within one representative experiment. \*p < 0.05 and \*\*p < 0.01, two-tailed t-test vs. 0 nM palbociclib or HuH-7 WT HuR. If not indicated otherwise, the differences were n.s.

Related to Figure 3.
<figure><img src="image_110.png" /><figcaption>**Figure S10.** (A) Quantification and representative histograms from flow cytometry analysis of MitoTracker Green FM staining in the WT HuR and K120/182R HuR SUMOylation mutant HuH-7 cells treated with 1 µM palbociclib for 3 days. (B) Segmentation masks of mitochondria (red) and ER (yellow) in electron microscopy images of WT and K120/182R HuR expressing HuH-7 cells. Scale bar is 1 µm. (C) Seahorse-based monitoring of the oxygen consumption rate (OCR) after treatment</figcaption></figure>
with oligomycin, carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP) and a
combination of rotenone and antimycin A in the HuH-7 cell lines stably expressing WT
HuR and the K120/182R HuR SUMOylation mutant. (D) Seahorse-based monitoring
of the OCR after treatment with oligomycin, FCCP and a combination of rotenone and
antimycin A, and quantification of mitochondrial respiration parameters in the HuH-7
cell lines stably expressing WT HuR and the K120/182R HuR SUMOylation mutant
subjected to a 3-day regimen with 1 µM palbociclib. (E) Representative images of
MitoSOX Red mitochondrial superoxide indicator staining in the WT and the
K120/182R HuR expressing HuH-7 cells. (F) Determination of total ATP content in the
WT and HuR SUMOylation mutant HuH-7 cells treated with 1 µM palbociclib for 3
days. (G) Representative curves and quantification of cytosolic Ca²⁺ levels with Fura-
2 AM fluorescent probe labelling after stimulating Ca²⁺ release from the ER with
thapsigargin and ATP, and addition of extracellular Ca²⁺ in the WT and SUMOylation
mutant HuR variant expressing HuH-7 cells treated with 1 µM palbociclib during 3
days.

(B and E) Images are representative of at least three biological replicates. (B) Scale bar is 1 µm.

(A, C, D, F and G) Data are represented as the mean ± SD of at least three biological replicates within one representative experiment. *p* < 0.05, **p* < 0.01, ***p* < 0.001 and ****p* < 0.0001, two-tailed t-test vs. 0 nM palbociclib or HuH-7 *WT HuR*. If not indicated otherwise, the differences were n.s.

Related to **Figure 5**.
<table><caption>**Table S1.** Summary of clinical data from a cohort of patients with HCC obtained from the Andalusian Biobank (Córdoba Node). Related to STAR Methods.</caption><tbody><tr><td>Patients [n]</td><td>86</td></tr><tr><td>Age, y [median (IQR)]</td><td>60.6 (64-67)</td></tr><tr><td>Etiology [n (%)]</td><td></td></tr><tr><td>HCV</td><td>30 (36.1)</td></tr><tr><td>Alcohol</td><td>21 (25.3)</td></tr><tr><td>HBV</td><td>11 (13.3)</td></tr><tr><td>Other</td><td>5 (6)</td></tr><tr><td>HCV + Alcohol</td><td>9 (10.8)</td></tr><tr><td>HBV + Alcohol</td><td>1 (1.2)</td></tr><tr><td>HCV + other</td><td>3 (3.6)</td></tr><tr><td>Alcohol + other</td><td>0 (-)</td></tr><tr><td>Histological differentiation [n (%)]</td><td></td></tr><tr><td>Well differentiated</td><td>30 (35.3)</td></tr><tr><td>Moderately differentiated</td><td>50 (58.8)</td></tr><tr><td>Poorly differentiated</td><td>5 (5.9)</td></tr><tr><td>Portal Hypertension [n (%)]</td><td>44 (5.2)</td></tr><tr><td>Microvascular invasion [n (%)]</td><td>33 (39.8)</td></tr><tr><td>Treated before surgery [n (%)]</td><td>23 (26.4)</td></tr><tr><td>Recurrence [n (%)]</td><td>39 (47)</td></tr><tr><td>Death [n (%)]</td><td>50 (61)</td></tr></tbody></table>
<table><caption>**Table S2.** Gene primer sequences used for qPCR and purchased from Sigma-Aldrich. Related to STAR Methods.</caption><thead><tr><th>Gene</th><th>Species</th><th>Forward primer</th><th>Reverse primer</th><th>Sequence</th></tr></thead><tbody><tr><td rowspan="2">ACTB</td><td rowspan="2">Homo sapiens</td><td>5'-ACTCTTCCAGCCTTCCTTCC-3'</td><td>5'-CAGTGATCTCCTTCTGCATC-3'</td><td></td></tr><tr><td>5'-GCCTCAAGATCATCAGCAATG-3'</td><td>5'-CTTCCACGATACCAAAGTTGT-3'</td><td></td></tr><tr><td rowspan="2">GAPDH</td><td rowspan="2">Homo sapiens</td><td>5'-CTTTGCTGACCTGCTGGAT-3'</td><td>5'-ATGTCCCCTGTTGACTG-3'</td><td></td></tr><tr><td>5'-ATCTGCTGCATCTGCTTG-3'</td><td>5'-CGACCTGGAAGTCCAACTAC-3'</td><td></td></tr><tr><td rowspan="2">RPLP0</td><td rowspan="2">Homo sapiens</td><td>5'-GGTCTGGACCAAAAGAAGAGGA-3'</td><td>5'-TCAGTGAAGCCATCTTTGGAGT-3'</td><td></td></tr><tr><td>5'-TCAGTAGACACCTCCCGTCTG-3'</td><td>5'-ACACCATCGACGTGTTCCAG-3'</td><td></td></tr><tr><td rowspan="2">SUMO1</td><td rowspan="2">Homo sapiens</td><td>5'-CGGGCCCTCTAGAAACTGTG-3'</td><td>5'-TCAGATTCCGATTTGGTGGG-3'</td><td></td></tr><tr><td>5'-CCTCCCGTAGGCTGTTGAAA-3'</td><td>5'-CTCACCATTCCCTGCCACCAT-3'</td><td></td></tr><tr><td rowspan="2">SAE1</td><td rowspan="2">Homo sapiens</td><td>5'-AGACAACTGGAGCCAGAAGC-3'</td><td>5'-CGCCTGGTATGTCTGTGTAAGA-3'</td><td></td></tr><tr><td>5'-ACTGTGCAGGCATGTGTAAC-3'</td><td>5'-CAAGACCCAGCTCAAGCAGA-3'</td><td></td></tr><tr><td rowspan="2">UBA2</td><td rowspan="2">Homo sapiens</td><td>5'-TGTGCTCGGACCCTTTTCTC-3'</td><td>5'-CGGACAGTGCGGAACTAAAG-3'</td><td></td></tr><tr><td>5'-ACGCCGATATCCACGAACTC-3'</td><td>5'-GGTGATGAGCCACCATCCAA-3'</td><td></td></tr><tr><td rowspan="2">PIAS1</td><td rowspan="2">Homo sapiens</td><td>5'-GTGCTTCCGTCCACTCTTGT-3'</td><td>5'-GGAGCTGGGCGAATTAAAGC-3'</td><td></td></tr><tr><td>5'-GCTGGTGGAGGCCAAAAACAT-3'</td><td>5'-GGGCTACAGTCAAACTGCAC-3'</td><td></td></tr><tr><td rowspan="2">SENP1</td><td rowspan="2">Homo sapiens</td><td>5'-CGAGCACGAGAAAGATTGCG-3'</td><td>5'-ACTGAATGTTCCCGCTCCTG-3'</td><td></td></tr><tr><td>5'-CTTTGCCTGGTCCCTCTAGC-3'</td><td>5'-GTGAGAGGCCTTCATGCACT-3'</td><td></td></tr><tr><td rowspan="2">SENP2</td><td rowspan="2">Homo sapiens</td><td>5'-CCGACCCTCTTTTGATGCCT-3'</td><td>5'-CAGCTGACTCCATCTTGGGG-3'</td><td></td></tr><tr><td>5'-TAACGCTGACAGAACCCAC-3'</td><td>5'-GCGGTTGCTACTCCCTTTCT-3'</td><td></td></tr><tr><td rowspan="2">SENP3</td><td rowspan="2">Homo sapiens</td><td>5'-TTCTGGAAGCTTTGGCTAGATCA-3'</td><td>5'-GCAGATTTGTCCCATCTTTATCTGT-3'</td><td></td></tr><tr><td>5'-CGGTTGCTACTCCCTTTCT-3'</td><td>5'-CTTCCAGTACTGTCCTCCTA-3'</td><td></td></tr><tr><td rowspan="2">SENP5</td><td rowspan="2">Homo sapiens</td><td>5'-CGGCGAGATGCATTCACAAG-3'</td><td>5'-TGACAGACGTACTGCTTGCT-3'</td><td></td></tr><tr><td>5'-CGGCGAGATGCATTCACAAG-3'</td><td>5'-GTTCCCTCACCCCTTCACAG-3'</td><td></td></tr><tr><td rowspan="2">SENP6</td><td rowspan="2">Homo sapiens</td><td>5'-GGCAGCTGATCAGAGGTTGT-3'</td><td>5'-GTCCTTCCAGCCTTCCTTCC-3'</td><td></td></tr><tr><td>5'-GTCCTTCCAGCCTTCCTTCC-3'</td><td>5'-GTCCTTCCAGCCTTCCTTCC-3'</td><td></td></tr><tr><td rowspan="2">SENP7</td><td rowspan="2">Homo sapiens</td><td>5'-CGGTTGCTACTCCCTTTCT-3'</td><td>5'-CGGTTGCTACTCCCTTTCT-3'</td><td></td></tr><tr><td>5'-CGGTTGCTACTCCCTTTCT-3'</td><td>5'-CGGTTGCTACTCCCTTTCT-3'</td><td></td></tr><tr><td rowspan="2">Gapdh</td><td rowspan="2">Mus musculus</td><td>5'-TTGATGGCAACAATCTCCAC-3'</td><td>5'-CGTCCCGTAGACAAAATGG-3'</td><td></td></tr><tr><td>5'-CGTCCCGTAGACAAAATGG-3'</td><td>5'-TCCCACTGTCCTCCTA-3'</td><td></td></tr><tr><td rowspan="2">Pias2b</td><td rowspan="2">Mus musculus</td><td>5'-TGACAGACGTACTGCTTGCT-3'</td><td>5'-CGGCGAGATGCATTCACAAG-3'</td><td></td></tr><tr><td>5'-TGACAGACGTACTGCTTGCT-3'</td><td>5'-CGGCGAGATGCATTCACAAG-3'</td><td></td></tr><tr><td rowspan="2">Senp1</td><td rowspan="2">Mus musculus</td><td>5'-CGGCGAGATGCATTCACAAG-3'</td><td>5'-GTTCCCTCACCCCTTCACAG-3'</td><td></td></tr><tr><td>5'-GTTCCCTCACCCCTTCACAG-3'</td><td>5'-GTCCTTCCAGCCTTCCTTCC-3'</td><td></td></tr><tr><td rowspan="2">Senp2</td><td rowspan="2">Mus musculus</td><td>5'-GTCCTTCCAGCCTTCCTTCC-3'</td><td>5'-GTCCTTCCAGCCTTCCTTCC-3'</td><td></td></tr><tr><td>5'-GTCCTTCCAGCCTTCCTTCC-3'</td><td>5'-GTCCTTCCAGCCTTCCTTCC-3'</td><td></td></tr></tbody></table>
<table><tr><td></td><td></td><td>Reverse primer</td><td>5'-ACAGGCCCAGCTTCCATAAC-3'</td></tr><tr><td rowspan="2">Senp3</td><td rowspan="2">Mus musculus</td><td>Forward primer</td><td>5'-TGGCAGAGGATGGGATGAGA-3'</td></tr><tr><td>Reverse primer</td><td>5'-GTCAGGGTCCAGAGGAGACT-3'</td></tr></table>
<table><caption>**Table S3.** Thermodynamic interaction parameters for T-rich DNA binding to HuR RRM1-2 species. *K*<sub>D</sub> stands for dissociation constant, Δ*H*, Δ*S* and Δ*G* are the thermodynamic parameters for the association process. Related to **Figure 2**.</caption><thead><tr><th>Titrant</th><th>Ligand</th><th>n</th><th>K<sub>D</sub> (μM)</th><th>ΔH (kcal/mol)</th><th>ΔG (kcal/mol)</th><th>TΔS (kcal/mol)</th></tr></thead><tbody><tr><td rowspan="4">T-rich DNA</td><td>HuR RRM1-2 WT</td><td>1.26 ± 0.04</td><td>1.68 ± 0.32</td><td>-15.27 ± 1.00</td><td>-7.88 ± 0.11</td><td>-7.39 ± 1.01</td></tr><tr><td>HuR RRM1-2 K120R</td><td>1.17 ± 0.04</td><td>1.64 ± 0.34</td><td>-13.51 ± 0.87</td><td>-7.89 ± 0.12</td><td>-5.62 ± 0.88</td></tr><tr><td>HuR RRM1-2 K182R</td><td>1.13 ± 0.06</td><td>1.73 ± 0.57</td><td>-15.94 ± 1.72</td><td>-7.86 ± 0.19</td><td>-8.08 ± 1.73</td></tr><tr><td>HuR RRM1-2 K120/182R</td><td>1.36 ± 0.04</td><td>2.57 ± 0.65</td><td>-12.07 ± 0.87</td><td>-7.62 ± 0.15</td><td>-4.44 ± 0.88</td></tr></tbody></table>
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