Fludarabine

Lipopolysaccharide (LPS) from Escherichia coli was purchased from DIFCO Laboratories (Detroit, MI). Rabbit anti-mouse p38 and ERK 1/2 mAbs, affinity-purified rabbit anti-phospho p-38, affinity purified mouse anti-phospho ERK 1/2, rabbit anti-total and phosphor-specific SAPK/JNK mAbs, rabbit polyclonal anti-STAT1, rabbit polyclonal anti-STAT3, and rabbit anti-phospho and total NF-κB mAb were purchased from Cell Signaling Technology (Danvers, MA). The p38 MAPK inhibitor 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB-203580), p42/44 ERK inhibitor 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U-0126), and JNK inhibitor anthra[1,9-cd]pyrazol-6(2H)-one, 1,9-pyrazoloanthrone (SP 600125) were purchased from Calbiochem (Mississauga, Ontario, Canada). Fludarabine (specific inhibitor of STAT-1) was obtained from Sigma-Aldrich (Mississauga, Ontario, Canada). STAT3 inhibitor, 2-Hydroxy-4-(4-methylphenyl) sulfonyloxy)acetyl)amino)-benzoic acid (S31-201) was obtained from Santa Cruz Biotechnology (Dallas, TX).

The LX-2 cells were treated with Fludarabine (50 μM, Sigma, USA) and incubated for 48 h. Proliferation of LX-2 cells treated with Fludarabine and MDI or without treated was detected by Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA) according to manufacturer’s instruction. For immunofluorescence studies, primary activated HSCs cultured for 1 month were grown on cover slips and treated with Fludarabine for 48 h or without, then incubated with the following antibodies: rabbit anti-αSMA (1:200, Abcam, MA, USA), mouse anti-Vimentin (1:500, Abcam, MA, USA). 20x pictures for each slip was analyzed using Image J software.

Recombinant IL-26, RANKL, and macrophage colony-stimulating factor (M-CSF) were purchased from R&D Systems (Minneapolis, MN, USA). SR11302 [activator protein 1 (AP-1) inhibitor], fludarabine (a STAT1 inhibitor), and parthenolide (an NF-κB inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA). LY294002 [a phosphoinositide 3-kinase (PI3K) inhibitor], SB203580 [a p38 mitogen-activated protein kinase (MAPK) inhibitor], PD98059 [an extracellular signal-regulated kinase (ERK) inhibitor], and AG490 [a Janus kinase (JAK)2 inhibitor] were obtained from Calbiochem (Schwalbach, Germany).

PDX models were established in 6-12 week old, male and female, NOD/Shi-SCID/IL-2Rγc^tm1sug/Jic (NOG), Mus musculus Linnaeus, 1758 (mouse) strain animals (in-house) as previously described [43 (link), 44 (link)].
Ibrutinib (Seleckchem; S2680) was resuspended in 1% methylcellulose and 0.4% Cremephor EL (Sigma; M0262 and C5135), and administered daily for 9 days by oral gavage at 12.5mg/kg, a dose which is sufficient to ensure 90% occupancy of Bruton tyrosine kinase (BTK) [45 (link)]. Rituximab (40mg/kg; Roche; 2530376), or saline control was given intravenously 3 times per week.[46 (link)] Fludarabine (0.625mg/kg, TEVA; 231-10-04151) and cyclophosphamide (6.25mg/kg, Baxter; 1001995501) or saline (control) were injected intraperitoneally 3 times per week, for two weeks, [47 (link)] whereas chlorambucil (5mg/kg; Sigma; CO253) or 3% dimethyl sulfoxide (DMSO, Sigma; D2650) vehicle control was administered daily for 5 days intraperitonally [48 (link)].
At specific time points (one week following treatment with chlorambucil, Fludarabine/cyclophosphamide or Rituximab and the next day following treatment with Ibrutinib) animals were sacrificed and single cell solutions produced from their homogenized spleens. CLL cells were sorted by flow-cytometry as demonstrated in the Supplementary Figure 1.

Fludarabine was purchased from Sigma‐Aldrich. Chk1 inhibitor SCH900776 (Merck, MWRCK KGaA, Darmstadt, Germany; MK‐8776) was kindly provided by K. Paruch (Department of Chemistry, Masaryk University). The inhibitor was dissolved as 100 μm stock solution and stored at room temperature (RT). Before use it was diluted in culture medium to 200 nm. A selective ATR inhibitor, VE‐821, was purchased from APIs Chemical Co., Ltd, Shanghai, China, KU55933, the ATM inhibitor, was from Tocris Bioscience (Ellisville, MO, USA) and NU7441 and the DNA‐dependent protein kinase inhibitor (DNA‐PKi) were from Axon Medchem (Groningen, the Netherlands). The inhibitors were dissolved in dimethyl sulfoxide as 10 mm aliquots and stored at −80 °C. The desired final concentrations were achieved by dilution with culture medium. The final concentrations were 10 μm for VE821 and KU55933, and 1 μm for NU7441.