Fig. S1 A. B. control Bortezomib. control Bortezomib. Rela ative expression N.D. MYC BCL2 XIAP TNF IL6

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Amalia Żukowska
5 lat temu
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1 Fig. S A. B. Rela ative expression control Bortezomib Rela ative expression control Bortezomib MYC BCL2 XIAP TNF IL6 N.D. MYC BCL2 XIAP TNF IL6 Figure S. Bortezomib inhibits NF- B signaling in human NSCLC cells. H222 (KRAS G2C ;TP53 C76F/Q6L ) (A) and H29 (KRAS G2A ;TP53 R273L ) (B) cell lines were treated with vehicle control or nm Bortezomib for 24 hours. NF- B target gene expression was measured by QPCR. Error bars are s.d. (n=3). N.D. denotes not detectable. Mutation information is from the Sanger COSMIC database.

2 Fig. S2 Re elative expression Myc Ccnd Bcl2 Bclxl 3TZ 3TZ LKR3 K LKR B KPA C KP L2D 5A 7B 832B KP 852B M Figure S2. QPCR analysis of NF- B target genes expression in murine lung adenocarcinoma cell lines. Values in the 3TZ fibroblast cell line is set to. Error bars are s.d. (n=3).

3 Fig. S3 A. Kras LSL-G2D/wt ;p53 flox/flox +Ade-Cre (2.5x 7 ) mg/kg wks microct IHC +Ade-Cre (2.5x 7 ) mg/kg per week 8 9 wks survival B. Kras LSL-G2D/wt +Ade-Cre (2.5x 7 ) mg/kg 2 wks microct +Ade-Cre (2.5x 7 ) mg/kg 25 wks IHC +Ade-Cre (2.5x 7 ) mg/kg gper week wks survival Figure S3. Bortezomib regimen in (A) KP and (B) K mice.

4 Fig. S4 Relative e tumor volume (fold) Vehicle Cisplatin Bortezomib p=. p= Time (days) Figure S4. Bortezomib reduces tumor volume in a subcutaneous tumor model. x 6 KP lung cancer cells were injected subcutaneously into NCR nu/nu mice. Tumor volume was quantified by caliper measurements. Treatments started when tumors reached ~mm 3 (set to in tumor volume axis and days in time axis). Arrows indicate Bortezomib (mg/kg) or Cisplatin (7mg/kg) injections. Error bars are s.d (n=4).

Fig. S5 A B ive viability Relat.6.4.2.8.6.4 sensitive resistant resistant 2 nm 5nM sensitive resistant resistant 2.2 5 Bortezomib (nm) Figure S5. Generating Bortezomib-resistant KP cell lines.

5 Fig. S5 A B ive viability Relat sensitive resistant resistant 2 nm 5nM sensitive resistant resistant Bortezomib (nm) Figure S5. Generating Bortezomib-resistant KP cell lines. (A) Relative cell viability in cell lines treated with increasing doses of Bortezomib for 24 hours. Cells lines were outgrown from Bortezomibresistant orthotopic tumors (resistant &2) or sensitive tumors (sensitive) as in Fig. 6C. Error bars are s.d. (n=3). (B) Colony formation assay in Bortezomib-resistant cells. 4 cells were plated in 6-well plate and treated with indicated drug concentration. Plates were stained 7 days later with crystal violet solution. Drug-containing medium was refreshed every 4 days.

6 Fig. S resistant+ctrl resistant+bz 24hrs pression Relative ex Bcl2 Bclxl Birc2 Birc5 Xiap Myc Ccnd Il6 Tnf Mmp3 Figure S6. Bortezomib-resistant KP cell lines showed down-regulation of NF- B targets regulating apoptosis and cell cycle upon drug treatment. A representative resistant cell line was treated with control or 5nM Bortezomib for 24 hours. Gene expression level was measured by QPCR and normalized to control-treated cells (set to ). Error bars are s.d. (n=3).

7 Fig. S7 A B sensitive resistant C N C N p sensitive resistant 5 75 c-rel p /p52 Relative nuclear NF- B activ vity p65 p52 p5 RelB c Rel 5 Parp Nemo Figure S7. Nuclear NF- B levels in Bortezomib-resistant and sensitive KP cell lines. (A) Nuclear (N) and cytoplasmic (C) fractions of protein lysates were immunoblotted with the indicated antibodies. Nemo and Parp serve as cytoplasmic and nuclear loading controls respectively.(b) Activity of NK- B in the nuclear extract was measured by ELISA assay. Values in the sensitive cell line is set to. Error bars are s.d. (n=3, p>.5 for all NK- B subunits).

8 Fig. S8 A p65 DAPI Merge sensitive resistant B p52/p DAPI Merge sensitive resistant Figure S8. Immunofluoresence in Bortezomib-resistant and sensitive KP cell lines. Fixed cells were stained with antibodies for p65 (A) and p52/p (B) (an antibody recognizing both the p52 and its precursor p) and Alexa-488 secondary antibody (green). Nucleus were stained with DAPI (blue). Images are 4x magnitude.

9 Fig. S9 Fold (Log2 ) 7 6 sensitive cell lines lines resistant cell lines p=. p=.3 p=.8 Bcl2 Bcl2l Birc2 Birc5 Xiap Myc Ccnd Il6 Tnf Mmp3 Figure S9. Transcriptional profile of NK- B targets in Bortezomib-resistant KP cell lines. Gene expression level was quantified by QPCR in four sensitive and four resistant KP cell lines. The average of sensitive cells is set to. p values indicate genes significantly different in resistant cells. Error bars are s.d. (n=4).

10 Fig. S A Re elative viability.4 LKR3 KP.2 KP2 KP3 KP B LKR3 KP Bay-782 CC3 Tubulin Bay-782 (μm) Figure S. Bay-782 induces cell death in KP cells. (A) Relative cell viability in cell lines treated with increasing doses of Bay-782 for 48 hours. (B) Bay-782 induces apoptosis in KP cells. Cells were treated with μm Bay-782 for 48 hours. Protein lysates were immunoblotted with cleaved caspase 3 (CC3) and Tubulin antibodies.

11 Fig. S A Control Bay-782 (48hrs) H&E cleaved caspase 3 B #CC3 + cells/m mm 2 tumor control Bay 782 Figure S. Bay-782 induces apoptosis in KP lung tumors. (A), H&E and cleaved caspase 3 (CC3) immunohistochemistry staining (2x) in vehicle control and Bay-782 treated KP lung tumors (mg/kg, 48hrs). (B) Quantification of CC3 positive cells. Error bars are s.d. (p<.5).

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