HEI-OC1 cells exposed to cisplatin exhibit the formation of SGs, while gentamicin causes blebbing of the cell membrane

HEI-OC1 cells were incubated with 60 µM cisplatin or 10 mM gentamicin for 24 h, compared to vehicle control. As a positive control for SG formation, HEI-OC1 cells were incubated with 0.5 mM arsenite for 30 min, compared to vehicle control. Immunocytochemistry was performed targeting main SG protein markers G3BP, PABP or TIA1, showing SG formation after exposure to cisplatin (Fig. 1). No SGs were observed in control experiments (data not shown). Gentamicin did not trigger the formation of SGs. Alternatively, HEI-OC1 cells exposed to gentamicin generated blebbing of the cell membrane (Fig. 1A), whereas control experiments showed no blebbing of the cell membrane (data not shown).

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FIGURE 1: Influence of arsenite, gentamicin, and cisplatin on the induction of SG formation in HEI-OC1 cells. (A) Representative images of HEI-OC1 cells incubated with 0.5 mM arsenite for 30 min, 10 mM gentamicin for 24 h, or 60 µM cisplatin for 24 h, fixed and stained with a dual antibody combination targeting SG proteins anti-TIA1/PABP and anti-G3BP. The arrows point to SGs. The arrow heads point to cell membrane blebbing formations. The scale bar is 10 µm. (B) Bar graphs depicting variations in the quantified number of SG per cell, dependent upon treatment with arsenite, gentamicin or cisplatin, compared to control. (C) Bar graphs illustrating the average size of SG in µm2, influenced by incubation with arsenite, gentamicin or cisplatin, versus control. On the bar graphs, the x-axis represents the treatment condition, and the y-axis represents whether the number of SG per cell, or the size of SG in µm2 by the mean ± SD of n = 3 independent experiments for all treatment groups. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test; with the adjusted p-value representing * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

HEI-OC1 cells incubated with cisplatin for 24 h generated on average 17.83 ± 0.733 SGs per cell, compared to vehicle-treated cells where no SGs were observed [p<0.001]. Exposure to arsenite for 30 min served as positive control for SG formation, with an average of 12.57 ± 5.733 SG per cell, compared to vehicle-treated cells where no SGs were observed [p<0.01] (Fig. 1B). The size of SGs formed after 24 h incubation with cisplatin exhibited an average size of 0.299 µm² ± 0.015 [p<0.05], while the size of SGs formed after 30 min of incubation with arsenite exhibited an average size of 1.745 µm² ± 0.204 [p<0.0001] (Fig. 1C).

Live cell imaging experiments were able to illustrate the onset of SG formation in HEI-OC1 cells exposed to cisplatin after 9 minutes incubations. Similarly, incubation of HEI-OC1 with arsenite elicited SG assembly, also initiating at the 9-minute treatment (Fig. 4, Suppl. Material S1A,B). During the 30-minute observation period following gentamicin treatment, no discernible formation of SGs was observed (data not shown).

Cisplatin induces the formation of SGs in H4 human neuroglioma cells

H4 cells were incubated with 40 µM cisplatin for 12 h or 10 mM gentamicin for 24 h, compared to vehicle control. As a positive control for SG formation, H4 cells were incubated with 0.5 mM arsenite for 30 min, and compared to vehicle control. Immunocytochemistry was performed targeting main SG protein markers G3BP, PABP or TIA1, showing cisplatin-induced SG formation (Fig. 2). No SGs were observed in control experiments (data not shown). Incubation of H4 cells with gentamicin did not trigger the formation of SGs (Fig. 2A). Cells incubated with cisplatin for 12 h displayed, on average, 14.94 ± 1.938 SG per cell, compared to vehicle-treated cells where no SGs were observed [p<0.0001]. Exposure to arsenite for 30 min was used as positive control for SG formation, with an average of 32.27 ± 4.118 SGs per cell, compared to vehicle-treated cells where no SGs were observed [p<0.0001] (Fig. 2B). The size of SGs formed after 12 h incubation with cisplatin was 0.348 µm² ± 0.341 [p<0.05], while the size of SGs formed after 30 min of incubation with arsenite exhibited an average size of 2.007 µm² ± 0.117 [p<0.0001] (Fig. 2C).

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FIGURE 2: Influence of arsenite, gentamicin, and cisplatin on the induction of SG formation in H4 human neuroglioma cells. (A) Representative images of H4 human neuroglioma cells incubated with 0.5 mM arsenite for 30 min, 10 mM gentamicin for 24 h, or 40 µM cisplatin for 12 h, fixed and stained with a dual antibody combination targeting SG proteins anti-TIA1/PABP and anti-G3BP. The arrows point to SGs. The scale bar is 10 µm. (B) Bar graphs depicting variations in the quantified number of SG per cell, dependent upon treatment with arsenite, gentamicin or cisplatin, compared to control. (C) Bar graphs illustrating the average size of SG in µm2, influenced by incubation with arsenite, gentamicin or cisplatin, versus control. On the bar graphs, the x-axis represents the treatment condition, and the y-axis represents whether the number of SG per cell, or the size of SG in µm2 by the mean ± SD of n = 3 independent experiments for all treatment groups. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test; with the adjusted p-value representing * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Live cell imaging experiments enabled the detection of SGs in H4 cells exposed to cisplatin after 3 minutes of incubation. Exposure to arsenite elicited SG assembly at 9-minute treatment (Fig. 4, Suppl. Material S1C,D). During the 30-minute observation period following gentamicin exposure, no discernible formation of SGs was observed (data not shown).

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FIGURE 3: Influence of arsenite, gentamicin, and cisplatin on the induction of SG formation in HEK-293T cells. (A) Representative images of HEK- 293T cells incubated with 0.5 mM arsenite for 30 min, 10 mM gentamicin for 24 h, or 15 µM cisplatin for 24 h, fixed and stained with a dual antibody combination targeting SG proteins anti-TIA1/PABP and anti-G3BP. The arrows point to SGs. The scale bar is 10 µm. (B) Bar graphs depicting variations in the quantified number of SG per cell, dependent upon treatment with arsenite, gentamicin or cisplatin, compared to control. (C) Bar graphs illustrating the average size of SG in µm2, influenced by incubation with arsenite, gentamicin or cisplatin, versus control. On the bar graphs, the x-axis represents the treatment condition, and the y-axis represents whether the number of SG per cell, or the size of SG in µm2 by the mean ± SD of n = 3 independent experiments for all treatment groups. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test; with the adjusted p-value representing * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

HEK-293T cells do not form SGs upon cisplatin or gentamicin treatment

HEK-293T cells were incubated with 15 µM cisplatin or 10 mM gentamicin for 24 h, compared to vehicle control. As a positive control for SG formation, cells were incubated with 0.5 mM arsenite for 30 min, and compared to vehicle control. Immunocytochemistry was performed targeting main SG protein markers G3BP, PABP or TIA1. We found that treatment with either cisplatin or gentamicin did not elicit SG formation in these cells (Fig. 3A). On the other hand, treatment with arsenite induced SG formation already after 3 minutes of incubation, compared to vehicle-treated cells, where no SGs were observed [p<0.05] (Fig. 4). After 30 min, the number of SGs exhibited an average of 2.023 ± 1.611 SGs per cell, when compared to the control group [p<0.05], and a size of 1.857 µm²
± 0.181 [p<0.0001] (Fig. 3B,C).

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FIGURE 4: Live cell imaging of HEI-OC1, H4 and HEK-293T cells with the G3BP marker following treatment with cisplatin compared to arsenite. Live cell imaging captures from (A) HEI-OC1 auditory cells; (B) H4 human neuroglioma cells; and (C) Human embryonic kidney HEK-293T cells subjected to treatment with cisplatin or arsenite representing maximal projections images of z-stacks at 3-minute intervals. Timepoint 0 was established when the treatment was administered and lasted for at least 30 min. Live confocal imaging with Airyscan was performed with a confocal point-scanning microscope (Zeiss LSM 800) using a 1.4 UPlanSApo 100x oil objective. The scale bar is 20 µm. The arrows point to SGs with G3BP marker. The yellow frames mark the timepoint at which the stress granules first become visible.

Cisplatin and gentamicin do not induce changes inthe levels of SG-related proteins

HEI-OC1, H4 and HEK-293T cell lines were subjected to SDS-PAGE after incubation with cisplatin for 12 or 24 h, gentamicin for 24 h, or arsenite for 30 min, compared to vehicle control. We analyzed key proteins implicated in the process of SG formation, including TIA1, PABP, G3BP1, and Caprin1, to assess expression changes after the specified treatment duration. Our results demonstrated stable protein levels of TIA1, PABP, G3BP1, and Caprin1 within HEI-OC1, H4, and HEK-293T cells following exposure to cisplatin or gentamicin in all our experiments. The exposure to arsenite for 30 min also did not change the levels of these proteins (Fig. 5).

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FIGURE 5: Detection of protein markers for SG formation in HEI-OC1, H4 and HEK-293T cells by SDS-PAGE immunoblot . Cell lysates (20 µg) of HEI-OC1, H4 and HEK-293T cells were subjected to SDS-PAGE after 12 or 24 h incubation with cisplatin or 24 h gentamicin, in comparison to 30 min of exposure to arsenite. After transfer, membranes were incubated with antibodies targeted against TIA1, PABP, G3BP1, and Caprin1, followed by incubation with an HRP-conjugated secondary antibody. Incubation with anti-Vinculin antibody served as loading control. Immunoblots were repeated three times as independent experiments. (A) HEI-OC1 cells incubated with 60 µM cisplatin for 24 h, 10 mM gentamicin for 24 h, or 0.5 mM arsenite for 30 min, compared to control. On the left, a specific section of the immunoblot membranes was selected to showcase untreated cells (-), versus cells subjected to treatment (+). On the right, the x-axis of the bar graphs represents the protein of interest on untreated cells (-), versus cells subjected to treatment (+), while the y-axis depicts the normalized protein signal with the mean ± SD. (B) H4 human neuroglioma cells incubated with 40 µM cisplatin for 12 h, 10 mM gentamicin for 24 h, or 0.5 mM arsenite for 30 min, compared to control. On the bar graphs, , the x-axis of the bar graphs represents the protein of interest on untreated cells (-), versus cells subjected to treatment (+), while the y-axis depicts the normalized protein signal with the mean ± SD. (C) HEK-293T cells incubated with 15 µM cisplatin for 12 h, 10 mM gentamicin for 24 h, or 0.5 mM arsenite for 30 min, compared to control. On the bar graphs, the x-axis of the bar graphs represents the protein of interest on untreated cells (-), versus cells subjected to treatment (+), while the y-axis depicts the normalized protein signal with the mean ± SD.

RNA is present in cisplatin-induced SGs in H4 cells

To assess the presence of RNA in SGs associated with cisplatin treatment, preparations from total H4 cell lysate, treated with 40 μM cisplatin for 12 h, were enriched for SG core by a series of centrifugation steps following the protocol, as previously described (Fig. 6). As a positive control, preparations from total cell lysates from H4 cells treated with 0.5 mM arsenite for 30 min were enriched for core; enrichment efficiency was evaluated using total protein and Western blotting analysis (Fig. 6B-D). We then assessed the presence of RNA in the purified SGs using Bioanalyzer and electropherogram representation of RNA separation from SG-enriched cisplatin-treated cells (Fig. 7). Our results confirmed the presence of RNA, as demonstrated by peaks at 26.52 nt and 27.25 nt, corresponding to 5S and 5.8S rRNA, as well as peaks at 42.50 nt and 49.85 nt length, corresponding to 18S and 28S rRNA (Fig. 7A,D). Total RNA preparations were treated with RNase to demonstrate the peaks were indeed RNA (Fig. 7B,D).

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FIGURE 6: Enrichment of SG cores from cells treated with cisplatin. (A) Schematic representation of the protocol for SG core enrichment adapted from Wheeler et al., 2017 [27] . (B) Western blot for G3BP1 in arsenite and cisplatin-treated H4 cell lysate (T) and their SG cores enriched fraction (EF). (C) Representative image of total protein amounts in T and EF from both arsenite and cisplatin-treated H4 cells. The relative amounts of total protein are shown at the bottom of each lane (D) Log2 plot of fold enrichment change in G3BP1 in EF relative to T.

Cisplatin-assembled SGs in cells persist after stress alleviation

Next, we assessed the dynamics of SGs formed in H4 cells formed upon 12 h exposure to 40 µM cisplatin. H4 cells were used given their increased susceptibility to cisplatin-induced cytotoxicity. The concentration and timepoint of cisplatin incubation was set prior to the alleviation experiments, as described. To further confirm the composition of the SGs formed in these conditions, we used two established SG markers, G3BP and Caprin1, and assessed the presence of SGs at 2, 6, and 12 h post cisplatin removal (Fig. 8A). The ratio of cisplatin-associated SGs per cell remained constant throughout (Fig. 8A,B). Alleviation experiments after arsenite-induced SG formation (0.5 mM arsenite for 30 min) served as control, and these experiments were performed in parallel to the cisplatin experiments, targeting the same proteins at the same time points, showing a complete clearance of SGs at 2 h post drug removal (Fig. 8A,B).

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FIGURE 7: Enrichment of SG cores from cells treated with cisplatin. (A) Schematic representation of the protocol for SG core enrichment adapted from Wheeler et al., 2017 [27] . (B) Western blot for G3BP1 in arsenite and cisplatin-treated H4 cell lysate (T) and their SG cores enriched fraction (EF). (C) Representative image of total protein amounts in T and EF from both arsenite and cisplatin-treated H4 cells. The relative amounts of total protein are shown at the bottom of each lane (D) Log2 plot of fold enrichment change in G3BP1 in EF relative to T.

DISCUSSION

Cell lines

Three cell lines were utilized in our study: the HEI-OC1 auditory cell line, the H4 human neuroglioma cell line (HTB-148; ATCC), and the human embryonic kidney cell line HEK-293T. The HEI-OC1 cells, derived from the cochlea of the Immortomouse^TM 65, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with L-alanyl-L-glutamine, 10% fetal bovine serum (FBS) and 0.3% penicillin at 33°C with 10% CO₂. The H4 human neuroglioma cells were cultured in Opti-MEM (Thermo Fisher Scientific) supplemented with L-alanyl-L-glutamine, 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO₂. Meanwhile, the human embryonic kidney cells HEK-293T were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO₂.

Drug treatments

Cells were plated onto 15-mm glass coverslips coated with 0.5% gelatin in 12-well plates at least 12 h before treatment initiation. 40,000 HEI-OC1 cells/well, 80,000 H4 cells/well, and 120,000 HEK-293T cells/well were seeded for experiments. HEI-OC1, H4 and HEK-293T cells were incubated with gentamicin (MilliporeSigma, Burlington, MA, USA) or cisplatin (MilliporeSigma) at various concentrations. Cisplatin and gentamicin were diluted in DMSO, which served as vehicle. Arsenite was diluted in deionized H₂O. The specific drug concentration chosen for experiments was stablished according to the concentration that induced 50% of cell death after 24 h of drug exposure, compared to vehicle control. This issue was addressed visually under light microscopy. Incubation with 0.5 mM sodium arsenite (MilliporeSigma) for 30 min served as positive control for SG formation. With gentamicin, the concentration chosen for experiments was 10 mM. For treatment with cisplatin, HEI-OC1 cells were incubated with 60 µM cisplatin for 24 h, and HEK-293T cells with 15 µM cisplatin for 24 h. An exception arose with H4 cells incubated with cisplatin, as no cells survived after a 24 h incubation time at any concentration. Consequently, the chosen cisplatin concentration was adjusted to an exposure time of 12 h, i.e., a concentration that induced 50% of cell death after 12 h of drug exposure, and compared to vehicle control. H4 cells were hence incubated with 40 µM cisplatin for 12 h, and compared to vehicle control. After treatment, cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature, and prepared for immunocytochemistry.

Immunocytochemistry

Cells were permeabilized with PBS containing 0.1% Triton X100 for 20 min and blocked with 1.5% normal goat serum for 1 h at room temperature, followed by incubation with a combination of two of these primary antibodies in the same blocking solution at a concentration of 1:1000 overnight at 4°C: mouse anti-G3BP (#ab56574, Abcam), rabbit anti-TIA1 (#SAB4301803, MilliporeSigma), rabbit anti-PABP (#ab21060, Abcam), rabbit anti-Caprin1 (#15112-1-AP, Proteintech). The following day, cells were incubated with blocking solution containing Alexa Fluor anti-rabbit 488 and Abberior anti-mouse 635P secondary antibodies at a concentration of 1:1000 for 2 h at room temperature. Shortly before mounting, a DAPI staining was performed for 15 min at room temperature. Coverslips were mounted on glass slides.

Confocal microscopy

Confocal images were acquired on an Abberior Expert Line STED microscope (Abberior, Göttingen, Germany) based on an Olympus IX83 inverted microscope using a 1.4 UPlanSApo 100x oil immersion objective. The excitation lasers used were 488 nm and 640 nm. Image stacks were acquired using the Imspector Software 16.3 (Abberior) with a 15 µs dwell time and a pinhole set at 1.1, xy pixel sizes of 80 x 80 nm and z-stack steps of 100-200 nm.

Live cell imaging

Cells were seeded at least 12 h previous to the experiments. 40,000 HEI-OC1, 80,000 H4, and 120,000 HEK-293T cells were plated in individual glass-bottom 35 mm µ-Dishes (Ibidi, Gräfelfing, Germany). The transfection was performed using the G3BP1-GFP plasmid utilizing Metafectene (Biotex, Munich, Germany) in a 1:2 ratio for HEI-OC1 and HEK-293T cells, and a 1:3 ratio for H4 cells, and according to manufacturer’s instructions 8 h for HEI-OC1 cells, and 24 h for HEK-293T and H4 cells. The culture medium was changed shortly previous treatment initiation. The plate was then transferred to the temperature- and CO₂-controlled chamber at the microscope setup, where treatment drugs were administered at the pre-determined concentrations, corresponding to timepoint 0 in the live cell imaging process. The live cell imaging was performed using a Zeiss LSM 800 with Airyscan confocal point-scanning microscope (Carl Zeiss, Jena, Germany) with a 1.4 UPlanSApo 100x oil objective. Z-stack images with 15 slices per image were captured at 3-minute intervals. Timepoint 0 was established when the treatment was administered, which lasted for a minimum of 30 min. At least three points on each plate were selected to monitor the progression of the effects of each treatment for every cell line. Confocal live cell images were captured under optimal conditions, settings were adjusted using the ZEN Software (Carl Zeiss).

Western blotting

Protein levels of TIA1, PABP, G3BP1 and Caprin1 were assessed by western blotting. After incubation, cells were lysed in protein extraction reagent lysis buffer (T-PER; Thermo Fisher Scientific) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentration was estimated using the Bradford method. Cell lysates were denaturized in 4x Laemmli’s sample buffer at 95°C for 5 min. 20 µg of protein were loaded and separated by SDS-PAGE. Gels were them immersed in 20% ethanol for 10 min, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific) for 7 min at 25 V, 0.9 A. PVDF membranes were subsequently placed in blocking solution containing 5% bovine serum albumin (BSA) in TBS-Tween 0.05% for 1 h at room temperature. Membranes were incubated overnight at 4°C in blocking solution containing the first antibody. The following primary antibodies were used: rabbit anti-G3BP1 1:6000 (#13057-2-AP, Proteintech), rabbit anti-TIA1 1:2000 (#12133-2-AP, Proteintech), rabbit anti-PABP 1:2000 (#ab21060, Abcam), rabbit anti-Caprin1 1:6000 (#15112-1-AP, Proteintech). Incubation of the same membranes with mouse anti-Vinculin 1:6000 (#sc-55465, Santa Cruz) served as a loading control. On the next day, membranes were washed in TBS-Tween 0.05% and incubated for 1 h at room temperature in blocking solution containing HRP-conjugated secondary antibodies 1:10,000. Chemiluminescent signals were characterized utilizing Fusion Fx Spectra 7.0 (Vilber, Marne-la-Vallée, France). Band intensity analysis was performed utilizing the Fiji open-source software 2.9.0.

Statistical analyses

SGs were quantified per number and/or size/area within cells using the channel corresponding to the G3BP staining, and analyzed semi-automatically using the Analyze Particle plugin from the Fiji open-source software 2.9.0. As SGs varied in size depending on drug exposure, SGs were counted by defining a specific size range specific to each drug. The pixel size (pixel^2) was set in a range between 0.10 – 10.0 for cells treated with cisplatin, and in a range between 0.70 – 10.0 for cells treated with arsenite or gentamicin. The statistical significance of the cell death, the formation of SGs per cell, and the average number of SG per cell ± SD for all three drugs in cells exposed to treatment versus untreated cells was carried out using one-way ANOVA with Tukey’s multiple comparison test. The adjusted p-value represented * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

SG core enrichment

H4 human neuroglioma cells were cultured to obtain a 90% confluence. Cells were treated with 40 μM cisplatin (MilliporeSigma) versus DMSO for 12 h or with 0.5 mM arsenite (MilliporeSigma) for 30 min versus H₂O. following which the cells were scraped, collected and centrifuged. The SG enrichment protocol was performed as described by Wheeler et al., 2017 27 . The cells were pelleted at 1500 g for 3 min at room temperature. The cell pellets were snap frozen and syringe lysis was performed using a 25G needle on ice in SG lysis buffer supplemented with ribonucleases inhibitors (Promega, Madison, WI, USA), as well as proteases inhibitors (Roche), containing 50 mM Tris HCl pH 7.4, 100 mM Potassium acetate, 2 mM Magnesium acetate, 0.5 mM DTT, 50 μg/ml Heparin, 0.5% NP40 and 1:5000 Antifoam B. Following lysis, the cell debris was pelleted at 1000 g for 5 min at 4°C. The supernatant was then used for a series of centrifugation steps as per the protocol to finally achieve a SGs-enriched pellet. The final pellet was resuspended in 60 μl SGs lysis buffer and centrifuged at 850 g for 2 min at 4°C followed by collection of the supernatant as the SG-enriched fraction.

Total protein detection and immunoblotting of SG core enriched fraction

Protein levels of G3BP1 in both total crude cell lysate (T) and SG core enriched fraction (EF) were estimated by western blotting. Protein concentrations were quantified using a Pierce BCA Protein Assay Kit and 10 µg of protein were loaded onto a 4-20% precast SDS-PAGE gel. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific) for 7 min at 25 V, 0.9 A. Immediately after transfer, the membrane was stained in Revert^TM Total Protein stain solution (Li-Cor, Bad Homburg, Germany) for 5 min at room temperature. Image for fluorescent immunodetection were acquired using Odyssey CLx (Li-Cor) according to the manufacturer’s instructions. After washing with ultrapure H₂O, the membrane was subsequently placed in blocking solution containing 5% BSA in TBS-Tween 0.05% for 1h at RT followed by overnight primary antibody (rabbit anti-G3BP1 1:6000, #13057-2-AP, Proteintech) incubation at 4°C. On the next day, membrane was washed in TBS-Tween 0.05% and incubated for 1h at room temperature in blocking solution containing an HRP-conjugated anti-rabbit secondary antibody 1:10,000. Chemiluminescent signals were characterized utilizing Fusion Fx Spectra 7.0 (Vilber). Total protein and bands intensity analysis, as well as the normalization of G3BP1 signal to the Total Protein signal in both T and EF, were performed manually utilizing the Fiji open-source software v2.14. The plot showing fold enrichment change in G3BP1 in EF relative to T was performed utilizing Graphpad Prism 8.4.3 software (Graphpad Software, Boston, MA, USA).

Total RNA isolation fromSG-enriched fractions

RNA isolation was performed using TRIzol^™ Reagent (#15596026, Thermo Fisher Scientific) following the manufacturer’s protocol. Briefly, equal volume of TRIzol^™ (60 μl) was added to SGs-enriched factions. The samples were incubated at room temperature for 5 min. 12 μl of chloroform was then added, and the samples were shaken and rested for 2-3 min at room temperature. The samples were then centrifuged at 14 000 g for 15 min at 4°C. The top clear phase containing RNA was carefully separated into a fresh microcentrifuge tube. RNA clean-up was performed by adding an equal volume of 100% ethanol and further using the RNA Clean and Concentrator 5^™ Kit (#R1014, Zymo Research, Irvine, CA, USA) following manufacturer’s protocol. RNA was finally eluted in 6µl DNase and RNase free water. This total RNA sample was then used for RNase degradation and rRNA depletion.

RNA degradation using RNase treatment

RNase degradation was performed by adding RNase A^™ DNase and protease-free (#EN0531, Thermo Fisher Scientific) to the RNA sample and incubating at 37°C for 2 h. The degraded RNA was cleaned using the RNA Clean and Concentrator 5^™ Kit (Zymo Research). Briefly, twice the amount of RNA binding buffer was added to the RNA sample, which was then thoroughly mixed before adding twice the amount of 100% ethanol. The clean-up was performed as per the manufacturer’s instructions.

Ribosomal RNA (rRNA) depletion from total RNA samples

Depletion of rRNA was performed utilizing the NEBNext^™ rRNA Depletion Kit (#E7400L, New England Biolabs, Ipswich, MA, USA). The protocol was performed as per the manufacturer’s instructions. NEBNext^™ RNAClean sample purification beads (New England Biolabs) were utilized to bind the remaining RNA. Finally, the RNA was eluted in 7 µl of DNase and RNase free water and then, 5 µl was collected for analysis.

Bioanalyzer RNA analysis

All the RNA samples including total RNA, degraded RNA and rRNA-depleted RNA were checked for their quality using Agilent 2100 Bioanalyzer^™ (Agilent Technologies, Santa Clara, CA, USA). Total RNA was assessed using the Agilent eukaryotic RNA Nano Kit (#5067-1511, Agilent Technologies) according to manufacturer’s instructions. Degraded RNA and rRNA-depleted samples were checked using the Agilent eukaryotic RNA Pico Kit (# 5067-1513, Agilent Technologies) as being more sensitive to lower RNA concentrations. The experiment was performed as per the manufacturer’s protocol. Electropherograms for each sample were obtained after each run.

Data availability

The authors state that all data necessary for confirming the conclusions presented in the manuscript are represented fully within the manuscript.