A Genetically Encoded Toolbox for Glycocalyx Engineering: Tunable Control of Cell Adhesion,

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1 TITLE A Genetically Encoded Toolbox for Glycocalyx Engineering: Tunable Control of Cell Adhesion, Survival, and Cancer Cell Behaviors AUTHORS Carolyn R. Shurer *, Marshall J. Colville *, Vivek K. Gupta, Shelby E. Head, FuiBoon Kai, Jonathon N. Lakins, Matthew J. Paszek * Contributed equally Number of pages: 8 Number of figures: 2 Number of supporting movies: 4 Supporting text full cloning details S1

2 Supporting Figure 1 Immunoblot A, Immunoblot showing the relative molecular weight of each of the Podxl mutants expressed transiently in HEK293T cells, n=2. Not that the 2/3 length Podxl is not detected by the anti-podxl antibody due to deletion of the recognition domain during cloning. S2

3 Supporting Figure 2 RTK Screen A, Full array image of PathScan RTK antibody slide. Induced cells were grown for 24 hours in either adherent or suspension conditions before being lysed and analyzed per the kit protocol. B, Schematic of RTK array slide. C, Key for schematic of RTK array slide. S3

4 SUPPORTING MOVIE LEGENDS Supporting Movie 1 - Time-course of Muc1 ΔCT expression. MECs were allowed to adhere and spread on fibronectin coated glass substrates before inducing expression of the fluorescent Muc1 ΔCT moxgfp variant. As fluorescence intensity increases cells begin to adopt a rounded phenotype and eventually detach from the substrate. Elapsed time is relative to the time of doxycycline addition (scale bar 100 µm). Supporting Movie 2 - Attachment followed by detachment. An MEC expressing cytoplasmic EGFP and Muc1 ΔCT drifts into the center of the frame, attaches to the substrate and spreads at 37 hours. After a period of attachment the cell rounds up and detaches from the substrate at 58 hours. Elapsed time is relative to the addition of doxycycline. Elapsed time is relative to the addition of doxycycline (scale bar 100 µm). Supporting Movie 3 Division leads to detachment. A MEC expressing cytoplasmic EGFP and Muc1 ΔCT divides at 37.5 hours following the addition of doxycycline. The two daughter cells detach from the substrate following division and float freely in the culture media. Elapsed time is relative to the addition of doxycycline (scale bar 100 µm). Supporting Movie 4 Attachment is required for division. A detached MEC expressing cytoplasmic EGFP and Muc1 ΔCT attaches to the substrate and spreads at 32.5 hours. At approximately 40 hours the cell divides, and both daughter cells detach from the substrate and float freely in the culture media. Elapsed time is relative to the addition of doxycycline (scale bar 100 µm). SUPPORTING TEXT S4

5 Cloning and Constructs Muc1 CT was inserted into a second-generation lentiviral vector with a doxycycline inducible promoter and puromycin resistance cassette as previously described 8. A lentiviral vector for stable, shrna-mediated knockdown of Muc1 was constructed by inserting the shrna sequence 5'- CCG GGA CAC AGT TCA ATC AGT ATA ACT CGA GTT ATA CTG ATT GAA CTG TGT CTT TTT G -3 into the plko.1 lentiviral backbone. The inducible PiggyBac transposon vector ppb teton was created first by PCR amplification and insertion of 5 and 3 PiggyBac ITRs from ppb 5 PTK3 (kindly provided by Dr. Alan Bradley, Welcome Trust Sanger Institute, UK) into a minimal backbone for bacterial propagation and selection consisting of ampicillin resistance gene and ColE1 origin of replication PCR amplified from pbluescriptii KS+ (Stratagene). Between ITRs a puromycin resistance expression cassette consisting of SV40 promoter puromycin acetyltransferase gene and SV40 polyadenylation sequence was then inserted back to back with a Tet regulatable expression cassette consisting of hybrid hepatamerized Tet operator minimal CMV promoter, chimeric synthetic intron between human β-globulin, and immunoglobulin heavy chain introns, multiple cloning site (MCS) and polyadenylation sequence from bovine growth hormone. To clone Muc1 CT into transposon vector, Muc1 N-terminus (fragment A) was amplified with PCR using a forward primer 5 - ACT ACT ACT AGT ACC ATG ACA CCG GGC ACC CAG T -3 and a reverse primer 5 - ACT ACT AGA TCT GGT CGT CGT CAT CCT TGT AAT CAG CA -3 using MMTV-Muc1 construct as the template (kindly provided by Sandra Gendler). The central sequence of Muc1 harboring tandem repeats (fragment B) was excised from the same construct using BglII and ApaLI. The C-terminus of the Muc1 CT (fragment C) was amplified using a forward primer 5 - ACT ACT GTG CAC AAC GGC ACC TCT GCC AGG GCT -3 and a reverse primer that introduced a stop codon immediately following the Muc1 transmembrane domain 5 - ACT ACT S5

6 GAA TTC CTA GCA CTG ACA GAC AGC CAA GGC AAT -3. The fragments A, B, and C were digested with SpeI/BglII, BglII/ApaLI, and ApaLI/EcoRI, respectively. These three fragments were subsequently cloned into SpeI/EcoRI-digested transposon vector. ppb teton SynMuc1 was prepared by inserting the DNA sequence 5 - TCA GGC ATA CTT TAT TGG CGA AAC CCA ACG GAA AGT GAT AGC ATC GTT TTG GCA ATT ATC GTC CCC AGT CTG CTC CTC TTG CTC TGC CTG GCT TTG TTG TGG TAC ATG CGC CGA CGA AGT ATG TAG GG -3 into the Bsu36I and EcoRI sites of ppb teton Muc1 CT. ppb teton Muc1 CT moxgfp was prepared by PCR of the moxgfp gene (Addgene plasmid # ) using the forward primer 5 - GGC ACC ATG GCA TGG TGT CCA AGG GCG AGG AGC TGT -3 and the reverse primer 5 - GGC ACC ATG GGC CTT GTA CAG CTC GTC CAT GCC GTG A -3 and non-directional insertion into the NcoI site of ppb teton Muc1 CT. For creation of ppb teton Podxl, the human Podxl cdna (clone ID , Dharmacon) was amplified via PCR with forward and reverse primers 5 - GGC AGG ATC CAC GAC ACG ATG CGC TGC GCG CT -3 and 5 - GGC AGA ATT CCT AGA GGT GTG TGT CTT CCT CCT C -3. The PCR product was subsequently inserted into the BamHI and EcoRI sites of ppb teton. ppb teton Podxl CT was generated through amplification of the Podxl cdna with 5 - GGC AGG ATC CAT GCG CTG CGC GCT GGC GCT CTC G -3 and 5 - CCG TCC TGA GGT TAG AGG CGC TGG TGG CAG CAG CCA TAG AGG -3 and insertion into the ppb teton vector as before. SynPodxl was created by amplification of the O- glycosylation rich ecto-domain from full-length human Podxl via PCR amplification with 5 - GGC AGG ATC CAT GCG CTG CGC GCT GGC GCT CTC G -3 and 5 - CCG TCC TGA GGT TAG CAT GCT GAA GCG GTC CTC GGC C -3 forward and reverse primers to add 5 BamHI and 3 Bsu36I restriction sites. Following restriction digest, the ectodomain was ligated S6

7 with the synthetic membrane proximal domain and 21-amino acid transmembrane domain into the ppb teton vector. Podxl CT and SynPodxl were subcloned into the BamHI and EcoRI sites of pcdna3.1(+) vector for transient expression and further cloning. Modular SynPodxl constructs were generated by introducing Bsu36I restriction sites between residues T40D41 and A179E180 (relative locations at the end of the signal peptide and 1/3 through the O-glycosylation rich region) by site-directed mutagenesis with the following primers: T40D41 forward 5 - CCT AAG GAC TCA TCT AAC AAA ACA GC -3, reverse 5 - CGT AGT AGT CTG GGT TGC -3 ; A179E180 forward 5 - CCT AAG GAA CAT CTG ACG ACC CCT -3, reverse 5 - TGC CTT AGT GGA TGT GAG -3. One mutant was generated for each new restriction site, and the resulting plasmids as well as pcdna3.1(+) SynPodxl were digested with Bsu36I and BamHI to yield 2 fragments from each mutant and the vector with transmembrane sequence from SynPodxl. 2/3 SynPodxl was constructed by ligation of the 129 bp fragment from T40D41 and 865 bp fragment from A179E180 into the vector. 4/3 SynPodxl was constructed in the same manner from the 537 bp fragment from A179E180 and 1.3 kbp fragment from T40D41. REFERENCES (1) Paszek, M. J.; DuFort, C. C.; Rossier, O.; Bainer, R.; Mouw, J. K.; Godula, K.; Hudak, J. E.; Lakins, J. N.; Wijekoon, A. C.; Cassereau, L.; et al. The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Nature 2014, 511 (7509), DOI: /nature S7

8 (2) Costantini, L. M.; Baloban, M.; Markwardt, M. L.; Rizzo, M.; Guo, F.; Verkhusha, V. V.; Snapp, E. L. A palette of fluorescent proteins optimized for diverse cellular environments. Nat. Commun. 2015, 6, 7670 DOI: /ncomms8670. S8