HepR21 cells have increased resilience to nutrient deprivation compared to HepG2 cells.

<p>[A] <b><i>Cell survivability assay</i></b><b>—</b> Effect of nutrient starvation on HepG2 and HepR21 cells was studied by treatment with nutrient deficient media, EBSS for 6, 12, 24 and 36 h. A set of cells were kept as control. The survivability of HepR21 cells was found to remain unaffected while HepG2 cells were highly sensitive upon prolonged starvation with an overall 50% decrease in growth after 36 h of starvation. [B] <b><i>Assay for ROS</i></b><b>—</b> ROS assay performed after the aforementioned treatments showed an increased ROS generation of 1.7 fold for 6 h to 3.6 fold for 36 h of nutrient starvation, found to be highly significant for 12 to 36 h of treatment for HepG2 (**). While significant increase in ROS of 2.2 to 2.5 fold in case of HepR21 cells (*) compared to untreated HepG2 cells was observed for 24 to36 h of nutrient starvation (*p<0.05 and **p<0.005, n = 3). [C] <b><i>Decline in endogenous HA and HA cables with increased period of nutrient starvation</i></b><b>—</b> Untreated and nutrient deprived HepG2 and HepR21 cells were immunocytochemically stained with commercial biotinylated HABP, then subsequently with Streptavidin conjugated to Alexa Fluor 430 and DAPI. Decreased levels of HA and HA cables with increased nutrient starvation was observed, more prominently in HepG2 cells while decrease in HA cables in HepR21 was observed after prolonged treatment only. Scale bar represents 10μ. [D] <b><i>Increased vacuole frequency upon amino acid deprivation in HepG2 compared to HepR21</i></b><b>—</b> Amino acid deprived media, EBSS, induced progressively increased generation of vacuoles in HepG2 with increasing period of nutrient deprivation (6, 12, 24 and 36 h) compared to untreated cells. Almost 10 fold increase in vacuole frequency in HepG2 cells was observed after 36 h of nutrient deprivation, while for the same treatment the HepR21 cells showed a mere 3 fold increase as compared to the untreated control cells. Statistical analysis using ANOVA with difference at a level of <b>*</b>p≤0.05 between groups considered as significant, revealed significant increase in vacuoles for 12, 24 and 36 h of nutrient starvation in HepG2 (*); while the increase was only significant after 36 h of EBSS treatment for HepR21, denoted as <b>#</b> in the figure.</p

Downregulation of autophagic marker MAP-LC3 in HepR21.

<p>[A] <b><i>HepR21 cells show decreased expression of MAP-LC3 in immunoblots</i></b><b> —</b> Immunoblotting of HepG2 and HepR21 cells grown for various time periods clearly depicts a downregulated expression of the autophagic marker MAP-LC3 in HepR21 cells. <b>[B] </b><b><i>Significant downregulation of MAP-LC3 expression in HepR21</i></b><b>—</b> Fold change analysis after normalization with the loading control GAPDH using ImageJ and further statistical analysis by ANOVA indicates significant downregulation of total MAP-LC3 in HepR21 cells compared to HepG2 cells for all the time periods. Fold changes are represented as mean ± SD (n = 3, *p<0.05) <b>[C] </b><b><i>Immunocytochemistry also illustrates downregulation and punctate staining of MAP-LC3 in HepR21</i></b><b>—</b> Immunocytochemical analysis done using the antibody for MAP-LC3 and subsequent reprobing with anti-rabbit Alexa Fluor 546 and DAPI revealed decreased expression of the autophagic marker MAP-LC3 with HABP1 overexpression in HepR21 as compared to the non-transformed HepG2 cells grown for 36, 48 and 60 h. The characteristic punctate staining of MAP-LC3 was obtained in both the cell lines.</p

Downregulation of autophagic marker MAP-LC3 in HepR21.

<p>[A] <b><i>HepR21 cells show decreased expression of MAP-LC3 in immunoblots</i></b><b> —</b> Immunoblotting of HepG2 and HepR21 cells grown for various time periods clearly depicts a downregulated expression of the autophagic marker MAP-LC3 in HepR21 cells. <b>[B] </b><b><i>Significant downregulation of MAP-LC3 expression in HepR21</i></b><b>—</b> Fold change analysis after normalization with the loading control GAPDH using ImageJ and further statistical analysis by ANOVA indicates significant downregulation of total MAP-LC3 in HepR21 cells compared to HepG2 cells for all the time periods. Fold changes are represented as mean ± SD (n = 3, *p<0.05) <b>[C] </b><b><i>Immunocytochemistry also illustrates downregulation and punctate staining of MAP-LC3 in HepR21</i></b><b>—</b> Immunocytochemical analysis done using the antibody for MAP-LC3 and subsequent reprobing with anti-rabbit Alexa Fluor 546 and DAPI revealed decreased expression of the autophagic marker MAP-LC3 with HABP1 overexpression in HepR21 as compared to the non-transformed HepG2 cells grown for 36, 48 and 60 h. The characteristic punctate staining of MAP-LC3 was obtained in both the cell lines.</p

HAS inhibition in HepR21 leads to ROS surge, decline in HA level and upregulated MAP-LC3 levels.

<p>[A] <b><i>Inhibition of HA synthesis led to increased generation of ROS</i></b><b>—</b> HepR21 cells subjected to treatment with different concentrations of HA synthetase inhibitor, 4-MU and untreated cells were assayed for ROS. 4-MU treatment resulted in excess generation of ROS as a function of time and concentration. The assay showed a ROS fold increase of 1.5 to 3 with increase in 4-MU concentration from 0.25–2 mM upon treatment for 6 h, with the increase being significant (*) for 2 mM of 4-MU treatment. Statistical analysis indicated highly significant increase in ROS from 0.50 (∼4 folds) to 2 mM (∼6.3 folds) for 12 h of 4-MU treated cells compared to untreated HepR21 cells, denoted by **. [B] <b><i>Decline in HA levels</i></b><b>—</b> HepR21 cells earlier reported to have higher HA pool were subjected to the aforementioned treatments with 4-MU and immunocytochemically analyzed for HA. Immunocytochemical analysis using commercial Biotinylated HABP and then reprobing with streptavidin cy3 and DAPI, illustrated decreasing level of HA with increasing concentration of 4-MU and the change to be more drastic for 12 h of treatment. The cells can be observed as individual entities, which were beforehand intricately linked with HA cables. Scale bar represents 10μ. [C] <b><i>Immunocytochemistry shows upregulation of MAP-LC3 upon inhibition of HA synthesis in HepR21 cells</i></b><b> —</b> Immunocytochemical detection of the autophagic marker MAP-LC3 revealed an increased expression after 6 h of 2 mM 4-MU treatment while it showed a prominent upregulation of the protein in the 12 h treated cells which were previously observed to generate excess ROS. [D-F] <b><i>Immunoblotting validates cytochemical analysis of upregulated MAP-LC3 levels</i></b><b> –</b> Immunoblots for MAP-LC3 of 4-MU treated (12 h) and untreated samples and further analysis as described previously revealed significant (*) upregulation of total MAP-LC3 and MAP-LC3-II for all the 4-MU treated samples compared to untreated HepR21 cells. This observation signifies induction of autophagy to be concomitant with HA depletion and ROS surge.</p

HA depletion leads to induction of autophagy and upregulation of tumor suppressors.

<p>[A-B] <b><i>Upregulated Beclin 1 in HepR21 upon decline in HA</i></b><b>—</b> Simultaneous immunoblotting of the 4-MU treated and untreated cells using anti-Beclin 1 and anti-GAPDH also revealed a significant (*) upregulation of the other autophagy marker and tumor suppressor, Beclin 1 upon 4-MU treatment in HepR21 cells. [C] <b><i>Immunocytochemical analysis for PTEN</i></b><b>—</b> HepR21 cells having increased tumor potency showed an upregulation of the autophagy modulator and tumor suppressor when subjected to 4-MU treatment suggesting a decline in tumor potency upon induction of autophagy.</p

HepR21 cells have increased resilience to nutrient deprivation compared to HepG2 cells.

<p>[A] <b><i>Cell survivability assay</i></b><b>—</b> Effect of nutrient starvation on HepG2 and HepR21 cells was studied by treatment with nutrient deficient media, EBSS for 6, 12, 24 and 36 h. A set of cells were kept as control. The survivability of HepR21 cells was found to remain unaffected while HepG2 cells were highly sensitive upon prolonged starvation with an overall 50% decrease in growth after 36 h of starvation. [B] <b><i>Assay for ROS</i></b><b>—</b> ROS assay performed after the aforementioned treatments showed an increased ROS generation of 1.7 fold for 6 h to 3.6 fold for 36 h of nutrient starvation, found to be highly significant for 12 to 36 h of treatment for HepG2 (**). While significant increase in ROS of 2.2 to 2.5 fold in case of HepR21 cells (*) compared to untreated HepG2 cells was observed for 24 to36 h of nutrient starvation (*p<0.05 and **p<0.005, n = 3). [C] <b><i>Decline in endogenous HA and HA cables with increased period of nutrient starvation</i></b><b>—</b> Untreated and nutrient deprived HepG2 and HepR21 cells were immunocytochemically stained with commercial biotinylated HABP, then subsequently with Streptavidin conjugated to Alexa Fluor 430 and DAPI. Decreased levels of HA and HA cables with increased nutrient starvation was observed, more prominently in HepG2 cells while decrease in HA cables in HepR21 was observed after prolonged treatment only. Scale bar represents 10μ. [D] <b><i>Increased vacuole frequency upon amino acid deprivation in HepG2 compared to HepR21</i></b><b>—</b> Amino acid deprived media, EBSS, induced progressively increased generation of vacuoles in HepG2 with increasing period of nutrient deprivation (6, 12, 24 and 36 h) compared to untreated cells. Almost 10 fold increase in vacuole frequency in HepG2 cells was observed after 36 h of nutrient deprivation, while for the same treatment the HepR21 cells showed a mere 3 fold increase as compared to the untreated control cells. Statistical analysis using ANOVA with difference at a level of <b>*</b>p≤0.05 between groups considered as significant, revealed significant increase in vacuoles for 12, 24 and 36 h of nutrient starvation in HepG2 (*); while the increase was only significant after 36 h of EBSS treatment for HepR21, denoted as <b>#</b> in the figure.</p

HepR21 cells have higher resistance to glutathione depletion compared to HepG2 cells.

<p>[A] <b><i>Significant surge in reactive oxidant level in HepG2 with exposure to BSO in a concentration dependent manner</i></b><b>—</b> HepG2 and HepR21 cells were treated with increasing concentrations of BSO (0.25 mM, 0.50 mM, 1 mM and 10 mM) for 24 h. ROS assay of the treated and untreated cells was performed and the fold change in ROS of the treated cells was calculated against untreated HepG2 cells taken as control. Progressive increase in ROS levels with increase in concentration of BSO in HepG2 starting from 1.5 to 3.25 folds was observed. On the contrary, HepR21 cells showed negligible increase in ROS levels initially compared to untreated HepG2 cells upon exposure to lower concentrations of BSO. Unpaired t-test using GraphPad indicated significant increase in ROS in HepG2 cells from 0.50 mM BSO treatment compared to the untreated HepG2 cells. The surge in ROS in HepG2 cells was highly significant for treatment with 1 mM and 10 mM BSO. Compared to HepG2 cells, HepR21 cells showed only a slightly significant rise in ROS levels for 1 mM and 10 mM BSO with respect to (w.r.t.) untreated HepG2 cells. Difference at a level of *p<0.05 and **p<0.005 between groups were considered as statistically significant. [B] <b><i>Survivability of both HepG2 and HepR21 cells remains unaffected</i></b><b>—</b> Cell survivability assay performed for the abovementioned treatments indicated no detrimental effect on both the cell lines.</p

Morphology of HepG2 and HepR21 remains unaffected on progression of growth with both cell lines showing redox insensitivity.

<p>[A] <b><i>Haematoxylin-Eosin Staining</i></b><b>—</b> Morphological analysis by H-E staining of HepG2 and HepR21 cells illustrated that HABP1 transformed HepG2 i.e. HepR21 although morphologically varied than its normal counterpart having a higher cell volume, with a more flattened spread out appearance showed no change in morphology as a function of time. Increased generation of vacuolated HepR21 cells upon progression of time point was not observed. A magnified view of both the cells for 36 h of growth has been shown. [B] <b><i>MDC staining shows absence of autophagic vacuolation in both HepG2 and HepR21 cells</i></b><b>—</b> HepG2 and HepR21 cells were grown for different periods starting from 36 to 84 h and subjected to MDC (0.05 mM) staining as per the protocol mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103208#s2" target="_blank">Methods</a>. Subsequent fluorescence microscopy indicated no positive staining for autophagic vacuoles for any point of growth between 36 h to 84 h. MDC staining of F-HABP07 cells was taken as positive control. Scale bar represents 10μ. [C] <b><i>HepG2 and HepR21 cells are both insensitive to external redox stimuli</i></b><b>—</b> ROS assay performed on HepG2 and HepR21 cells after prior treatment of cells with varying concentration of H₂O₂ (0 µM, 50 µM, 100 µM, 250 µM and 500 µM) for one hour indicated both HepG2 and HepR21 cells to be redox insensitive even on exposure to 500 µM of H₂O₂. [D] <b><i>H₂O₂ treatment has no effect on survivability of HepG2 and HepR21 as evident from MTT Assay</i></b><b>—</b> HepG2 and HepR21 cells were grown in complete media till 48 h and then treated with the abovementioned concentrations of H₂O₂ for 1 h. Viability assay performed thereafter revealed that even 500 µM of H₂O₂ has no effect on the survivability of both the cell lines. The media was not changed at any point.</p

Downregulation of tumor suppressor PTEN upon HABP1 overexpression in HepG2.

<p>[A-B] <b><i>Downregulation of expression of the autophagic modulator PTEN in HepR21</i></b><b>—</b> Autophagic modulator and also a known tumor suppressor PTEN was observed from immunoblots to have a significantly reduced expression (∼30%) in HepR21 cells compared to HepG2 after 60 h of growth, indicating a higher tumorigenicity of HepR21. Fold changes were calculated taking GAPDH as loading control, using ImageJ and expressed as mean ± standard deviation (SD) of observations in triplicate (n = 3). Statistical analysis of significance was done by Single factor one-way ANOVA (*p<0.05). [C] <b><i>Immunocytochemical analysis corroborates immunoblot findings</i></b><b> —</b> Decreased expression of PTEN was also detected in HepR21 cells at 60 h of growth compared to that in HepG2 from cytochemical analysis.</p

Upregulated MAP-LC3-II, differential expression and localization of tumor suppressor p14ARF in HepG2 unlike HepR21 upon nutrient deprivation.

<p>[A-E] <b><i>Elevated levels of total MAP-LC3 and MAPLC3-II in HepG2 upon nutrient starvation</i></b><b>—</b> After the aforementioned treatment with EBSS, the HepG2 and HepR21 cell lysates were immunoblotted with MAP-LC3 (1∶1000) and GAPDH (1∶20,000) antibodies. The fold change in expression of MAP-LC3 was calculated after normalization with GAPDH expression by IMAGE J and subsequently analyzed using ANOVA and represented in the graph as mean ± SD. The analysis showed a progressively increased expression of total MAP-LC3 in HepG2 along with increased period of starvation, significant for all the treatments compared to the untreated HepG2 cells. The expression of MAP-LC3-II (16 kDa), the lipidated form of MAP-LC3, indicative of the amount of autophagy taking place, was also found to be significantly upregulated for the abovementioned exposure to EBSS in HepG2 cells. Statistical analysis revealed a significantly increased expression of total MAP-LC3 and MAP-LC3-II only after 24 h of nutrient starvation in HepR21 cells (<b>*</b>p<0.05, n = 3). [F] <b><i>Cytochemical expression of autophagic marker MAP-LC3 upon nutrient starvation</i></b><b>—</b> As observed in the immunoblot, an augmented expression of the autophagic marker MAP-LC3 was observed in HepG2 cells by sequential probing with anti-MAP-LC3, anti-rabbit Alexa Fluor 546 and DAPI on fixed, treated and untreated cells. While the HepR21 cells showed the characteristic punctate staining throughout with only a slight rise after prolonged nutrient starvation. [G] <b><i>Differential expression and localization of the tumor suppressor p14ARF observed for HepG2 and HepR21</i></b><b>—</b> A comparative immunocytochemical analysis of the expression of the tumor suppressor protein, p14ARF in HepG2 and HepR21 indicated an elevated expression with the increase in period of nutrient deprivation in both the cell lines, more prominently in HepG2. Interestingly, a prominent differential localization of the protein in the two cell lines was also noticed. While the protein was predominantly expressed in the cytoplasm in HepG2 cells, it was observed to be translocated to the nucleus progressively with nutrient deprivation in case of HepR21 cells.</p