TCF3 expression depends on SHMT2 function. (A) Integration of CRISPR/Cas9 drop out screen results (CSS, CRISPR screen score) in BL60 cells with total proteome changes (SILAC log fold change [LFC]) in inducible SHMT2 knockdown BL60 cells (shSHMT2#2) on day 5 after induction of shRNA expression. Selected gene names/proteins are indicated. (B) Schematic illustration of the functional role of TCF3 in BL adapted from Schmitz et al. Indicated mutation frequencies were obtained from Schmitz et al, 2012; Love et al, 2012; Grande et al, 2019; Bouska et al, 2017; López et al, 2019.^11,25-28 (C) Representative western blot shows inducible TCF3 knockdown in BL60 cell line on day 5 after induction of shRNA expression. GAPDH served as loading control (n = 3; Student t test, shCtrl vs shTCF3.1, P = .003; shCtrl vs shTCF3.2, P = .017; shCtrl vs shTCF3.3, P = .005). (D) Coculture experiment with BL60 TCF3 doxycycline-inducible knockdown cells (3 different shRNA constructs) compared with a nontargeting control shRNA (shCtrl). Cells were cocultured with wild-type cells and analyzed by flow cytometry using the GFP reporter of the vector. GFP-positive cells were normalized to day 1 upon induction (n = 3, mean ± SEM; P < .001 in two-way ANOVA). (E) Cell cycle analysis of the BL60 cell line expressing an inducible shRNA against TCF3 (shTCF3#3) or a nontargeting control shRNA (shCtrl; n = 3, mean ± SEM; **P < .01 in Student t test, ***P < .001 in Student t test). (F) Representative western blots of lysates derived from BL60 and Ramos cells at day 8 upon SHMT2 knockdown (n = 3-4; TCF3 [BL60] P = .02 [Ramos] P = .002, SHP-1 [BL60] P = .02 [Ramos] ns). GAPDH served as loading control. (G) Western blots show SYK Tyr525/526 and AKT Ser473 phosphorylation levels upon inducible knockdown (shTCF3#3) in BL60 cell line 48 hours after induction of shRNA expression compared with a nontarget control shRNA (shCtrl). GAPDH served as loading control. Phosphorylation levels were normalized to SYK or AKT expression, respectively (n = 3; P < .04 in Student t test for pSYK). (H) TCF3 reconstitution using a retroviral vector with GFP fluorescent reporter in constitutive SHMT2 knockdown cells (shSHMT2#1) with an RFP fluorescent reporter in BL60 cell line. Double-positive (GFP/RFP) cells were followed up in flow cytometry (n = 4, mean ± SEM; *P < .05 in Student t test). (I) Phosphoproteomic SILAC-based LC-MS analysis in inducible SHMT2 and CD79a knockdown vs control BL60 cells upon doxycycline induction. SILAC ratios are average of 2 replicates. Differentially phosphorylated sites were defined as phosphosites quantified in at least 2 replicates exhibiting an absolute log2 SILAC ratio >0.5. The Pearson’s correlation coefficient is shown. P value is from a Pearson’s correlation test. (J) SILAC log2 fold change (LFC) for BCR effector protein expression and tyrosine phosphorylation of the indicated BCR effectors upon inducible SHMT2 knockdown vs control in BL60 cells on day 5 after induction of shRNA expression. Differentially phosphorylated phosphosites (Benjamini-Hochberg adjusted P value < 1e-3) are colored in red. SILAC ratios are average of 3 and 2 biological replicates for total proteome and phosphoproteome, respectively. (K) Cell viability (XTT assay) of BL60 cells expressing either a constitutively active variant of the catalytic PI3K subunit P110α (MP110*) or the empty vector (control) that were treated with SHIN1 at a final concentration of 2 µM or dimethyl sulfoxide (DMSO) at day 0. Background corrected absorbance is shown (n = 4, mean ± SEM; on day 4 control + DMSO vs control + SHIN1, P < .001; control + DMSO vs MP110* + DMSO, P < .01; MP110* + DMSO vs MP110*+ SHIN1 ns in Bonferroni posttest).