Fig 2: Signaling analysis of THP-1 (A) MV4-11 (B) and 32D FLT3 ITD cells expressing TMD mutant PTPRJ. (A) THP-1 PTPRJ KO cells or cells re-expressing PTPRJ wt, G979L, or G983L were starved for 4 h in serum-free medium, then stimulated with 200 ng/ml FLT3 ligand for 5 min (+FL) or left unstimulated (–FL) and lysed in RIPA buffer. (B, C) MV4-11 and 32D FLT3 ITD PTPRJ KO cells or cells re-expressing PTPRJ wt, G979L, G983L, or G987L were starved for 4 h in serum-free medium and treated with DPI (0.5 µM), AC220 (20 nM), or mock (DMSO), as indicated, then lysed in RIPA buffer. Equivalent amounts of protein samples were separated by SDS-PAGE and blotted to a nitrocellulose membrane. Blots were first probed by phospho-site specific antibodies recognizing pSTAT5 (Y694), pAKT (S473), and pERK1/2 (T202/Y204). Blots were re-probed for total STAT5, AKT, and ERK1/2 and subsequently analyzed with antibodies recognizing vinculin (124 kDa) and beta-actin (42 kDa) as a loading control. Left: Representative immunoblots are shown. Dashes indicate positions of molecular weight standard bands. 79 – G979L; 83 – G983L; 87 – G987L Right: Quantification of specific phosphorylation of AKT, ERK1/2, and STAT5 in relation to the total protein level. Values were normalized to FL-stimulated or DPI treated wt and are given as mean ± standard deviation, n = 3 - 4. Statistics: two-way ANOVA and Dunnett’s multiple comparisons test; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 compared to respective wt.

Fig 3: Clonal growth of FLT ITD cells expressing TMD mutant PTPRJ proteins. MV4-11 and 32D FLT3 ITD PTPRJ KO cells re-expressing PTPRJ wt, G979L, G983L, or G987L were seeded in triplicates in 1.27% methylcellulose-containing, cytokine-free medium. Colonies were stained with iodonitrotetrazolium chloride after 7 days (32D FLT3 ITD) or 10 days incubation (MV4-11) and quantified using Image (J) (A) Number (#) of colonies per well relative (rel) to wt is shown. Values are given as mean ± standard deviation, n = 3. Statistics for MV4-11: one-way ANOVA and Dunnett’s multiple comparisons test; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to respective wt (ns; not significant). Statistics for 32D FLT3 ITD: unpaired two-tailed t-test; * p ≤ 0.05. (B) Representative images of whole wells are shown. 79 – G979L; 83 – G983L; 87 – G987L.

Fig 4: Phosphorylation of FLT3 in MV4-11, THP-1, and 32D FLT3 ITD PTPRJ KO AML cell lines stably re-expressing PTPRJ wt or TMD G979L, G983L, or G987L mutants. Cells were starved for 4 h in serum-free medium and lysed in RIPA buffer. During starvation, MV4-11 and 32D FLT3 ITD cells were treated with DPI (0.5 µM) or AC220 (20 nM), as indicated. THP-1 cells were stimulated with FL (200 ng/ml, 5 min) before harvest. FLT3 was enriched from 100 – 180 µg lysate by wheat germ agglutinin precipitation and subjected to SDS-PAGE and immunoblotting. Blots were first probed by phospho-site specific antibodies recognizing FLT3 pY591 and re-probed for total FLT3. (A–C) Left: Representative immunoblots (n = 3) are shown. Positions of immature, high mannose (HM, 130 kDa) and mature, complex glycosylated (CG, 160 kDa) forms of FLT3 are indicated by arrows. Positions of 130 and 180 kDa molecular weight standard bands are shown on the left side of the blots. 79 – G979L; 83 – G983L; 87 – G987L. Right: Quantification of specific FLT3 phosphorylation. Values were calculated as the ratio of phosphorylated to total receptor (sum of HM and CG signal) and normalized to DPI-treated or FL-stimulated wt. All values are given as mean ± standard deviation, n = 3. Statistics: one-way ANOVA, followed by Dunnett’s multiple comparisons tests; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 compared to wt.