Fig 2: Comparison of Isogenic HCT116 Cell lines with and without activated KRAS. (a) The parental CRC HCT116 (KRAS G13D/+) cell line has an activating mutation of KRAS. Here we compare this cell line to an isogenic HCT116 (-/+) cell line that has the mutated KRAS allele knocked out, leaving the cell with only one WT KRAS allele. PTPRS CRISPR KO and CRISPR control paired cell lines were made in both HCT116 parental (KRAS G13D/+) and the isogenic HCT116 (-/+) cell lines. Extracts prepared from growing cultures were used for western blot analysis of ERK, phospho-ERK, MEK, phospho-MEK, AKT andphospho-AKT. The HCT116 (-/+) is shown to have natively less phospho-ERK compared to its parental HCT116 cell line with the activated KRAS mutation. The KO of PTPRS for both cell lines shows a dramatic increase in phospho-ERK and phospho-AKT. Quantitations were determined by normalizing the phosphorylated protein values with the total protein; then dividing the PTPRS KO by the CRISPR control values. All experiments were done in triplicate. The mean and standard deviation are shown. Two-tailed, paired t test was used to determine the statistical significance for comparison as indicated. (b) Active Ras pull down for all cell lines. The mutant KRAS cells lines, HCT116 and SW620, show a constitutively active Ras. The WT KRAS PTPRS KO cell lines, KM12L4A and the isogenic HCT116 (-/+), show a reduced amount of active Ras. KM12L4A was also used for negative and positive controls incubating the samples with GDP and GTPs; HCT116 and HCT116 (-/+) samples were incubated with GTPs as a positive control.

Fig 3: Dasatinib treatments increased apoptotic response to ERK inhibition in HCT116 cells.Parental cells with PTPRS (labeled PTPRS +) were treated with ERKi and Dasatinib for 48 hours and compared to PTPRS KO cells (labeled PTPRS -) which were treated with ERKi alone for 48 hours. (A) PTPRS containing cells (lanes PTPRS +) labeled: untreated, Dasatinib (0.1 µM), ERKi SCH772984 (0.1 µM), and a combination of Dasatinib and ERKi. The Dasatinib treatment showed a reduction in phospho-SRC-Y419, but little change in phospho-ERK1/2-T202/Y204. Conversely, the ERKi treatment decreased phospho-ERK1/2-T202/Y204 but not phospho-SRC-Y419. The combination of both inhibitors was able to eliminate the phosphorylation of both ERK and SRC. This phosphorylation pattern matches that of the PTPRS KO cells when treated solely with the ERKi. Lane 8 shows the decrease in phosph-SRC in PTPRS KO cells treated with ERKi. The experiment was performed in triplicate for quantitative densitometry analysis see Supplementary Figure 5. (B) when both Dasatinib and ERKi were used in combination (lane 4), treatment of HCT116 + PTPRS revealed the induction of apoptosis equal to that induced by ERKi alone in PTPRS KO cells (lane 6). The paired, two-tailed t test was performed for comparisons. NS – not significant; ** p < 0.01; *** p < 0.001.

Fig 4: siRNA knockdown of SRC increased apoptotic response to ERK inhibition.HCT116 PTPRS KO and parental cells + PTPRS were treated with siRNA to SRC for 24 hours and then incubated with an ERKi for 48 hours. (A) Western analysis: Parental control cells (+PTPRS) treated with control siRNA (–), ERKi (SCH772984), siRNA to SRC (+), and a combination of SRC siRNA and ERKi; PTPRS KO (-PTPRS) cells were given the same treatments respectively. In the figure the two control lanes on the left were taken from the right end of this original blot and moved to conform to Figure 6 and for ease of understanding. The experiment was performed in triplicate for quantitative densitometry analysis see Supplementary Figure 6. (B) Annexin V apoptosis assays for these treatments revealed that the siRNA knockdown of SRC in combination with ERKi treatment in cells containing PTPRS caused the same amount of apoptosis as the ERKi in PTPRS KO cells. “116Ctl” represent parental cells + PTPRS, 116KO are HCT116 with PTPRS knockout cells, siC is siRNA scrambled control RNA, NT = not treated and ERKi = SCH 772984. The addition of the SRC siRNA 24 hours before a 48-hour ERK inhibition of the PTPRS KO cells did not enhance apoptosis (lane 6 compared to lane 8). The paired, two-tailed t test was performed for comparisons. NS – not significant; ** p < 0.01.

Fig 5: PTPRS affects the localization of ERK. (a) Immunofluorescent staining shows ERK nuclear localization in HCT116 PTPRS KO cells (upper left) and general whole cell staining in HCT116 control cells (lower left). A comparison of the control cells to the KO shows that the PTPRS KO cells have nuclei that are enriched with total ERK (arrow in top left compared to arrow in bottom left). The ERK phosphorylation in its location is confirmed with a MEK inhibitor (PD98509, middle panels). The inhibition of MEK prevent ERK phosphorylation resulting in ERK not being translocated to the nucleus (middle images have nuclei with very low-stained signals for ERK). Staining for phospho-ERK (right panels) shows a very dynamic difference with the PTPRS KO cells showing a bright signal and the control cells having very weak signal. Supplementary Fig. 4 shows the linear profile of fluorescence intensity for both DAPI and ERK/phospho-ERK. These measurements confirm the increased signal seen in the nuclei of PTPRS KO cells. (b) Western blot for the cells in Fig. 8a. The left two lanes are untreated HCT116 cells and right two are treated with 5 µM of MEK inhibitor PD98509 for 24 hours. The upper blot confirms the PTPRS knockout in the KO cells. The lower blots for phospho-ERK and total ERK confirm the increased phospho-ERK for the PTPRS KO in the left two lanes. The right most lanes confirm that the MEKi prevented the phosphorylation of ERK. (c) Multichannel blot of cells transfected with PTPRS with a C-terminal Flag tag. This blot uses both a PTPRS (rabbit green) and Flag (mouse red) antibody. Here the cleavage of PTPRS is illustrated. Full length PTPRS is 217 kDa (yellow), the N-terminal subunit containing extracellular and transmembrane domains are 140 kDa (green) the C-terminal Subunit containing phosphatase D1 and D2 domains are 78 kDa (red). This cleavage was a consistent result, and seen in all cell lines used. (d) Assessment of PTPRS transfection on ERK localization. HCT116 and KM12L4A PTPRS KO cells were transfected with a RFP C-terminal tagged PTPRS. Here we examined the localization of PTPRS (red) and total ERK (green) as well as their co-localization (orange). The PTPRS transfected cells (red) show a critically reduced level of ERK in their nuclei (white arrows) when compared to the cells not over expressing PTPRS (red arrows), which have bright green nuclei (ERK). (e) Western blotting corresponding to the cells used in 8d and 8f. The left three lanes show the PTPRS KO compared to the control cells and PTPRS KO cells transfected with PTPRS. The third lane shows that PTPRS transfected back into PTPRS KO cells reduces the increased phospho-ERK back to levels equivalent to the control cell line. The right three lanes show the ISP inhibited the transfected PTPRS activity allowing for increased phospho-ERK. (f) The nuclear reduction of ERK as a result of PTPRS transfection is reversed when PTPRS is inhibited by the ISP. HCT116 and KM12L4A PTPRS KO cells were transfected with the RFP tagged PTPRS and then treated with the PTPRS inhibitor ISP. The reduction in nuclear ERK (8d) is completely reversed (8f) when PTPRS is inhibited. Both the cells overexpressing PTPRS (white arrows) and non-transfected cells (red arrows) show bright ERK signal in their nuclei. Supplementary Fig. 5 shows the DAPI stains for these images. (g) Duo-Link In Situ staining for PTPRS and ERK co-localization. HCT116 PTPRS KO cells were transfected with a C-terminal FLAG tagged PTPRS or control empty vector. The cells were then labeled with a FLAG mouse Ab and an ERK rabbit Ab. The red dots indicate a successful duolink reaction, which requires both antibodies to be in close proximity. The PTPRS transfected cells show an ample amount of red signal (left), and the empty vector cells do not show a significant amount of signal (right). These data suggest a direct association between PTPRS and ERK.