Genome-scale CRISPR–Cas9 screen reveals novel regulators of B7-H3 in tumor cells


 LPS (0,25,50,100,125,175,200,250,300,400,500ng/ml),and H2O2 (0,50,150,250,500,800 μM). 
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) 
CD276-F CTGAGCCTGGCTTCAGCCTG CD276-R CAGAACACCTCAGCCTCAG SP20H-F1 GTTGATCTCCTAGAAAAATCTCAGG SP20H-R1 GCTTGTTATAGAGCAGTCTG SP20H-F2 GAGAAGCTTGTTATGCAAGAGAC SP20H-R2 TGCTATAGAAGGATCAAGACAGAG F118A-F CTCGTGGAGAAGCTGCATCG F118A-R AGGACCTTGGTCTTGTCCTTC EIF4E-F1 TTCATTCATCTGTTGATGG EIF4E-R1 AACTAATAAAGCATAATCACC EIF4E-F2 ATATTGCTGTCATAGATGTG EIF4E-R2 TGAACTCAAGACTCACAC MNK1-F1 TGCTGCTTTGCCAACAGAG MNK1-R1 TGTGGGGACAGGAGTGAG MNK1-F2 ACCATCAGTGTAAACCTCC MNK1-R2 CATCTGGCTCTAACTTGAC GTF3C4-F TGCCATCTCCTTGAGTAAATGC GTF3C4-R TGCAGTCTGTTGAGATTTGCC SRP72-F TGAATGTTTGGGTCATTTGG SRP72-R CTATGGAAAGCACAAATACTCC SRP9-F GCGTCGGTTGGCGACTC SRP9-R GCAGGGTCCATTTGTGTTCATTC SRPR-F GGCATTTTTGAGAGCATTCC SRPR-R CTGACCTTGGCAAGATTAGTAG SRP14-F CGTTACTCGAGACCTGTGC SRP14-R GAATGTGCTCAGTACAGG SRP19-F ACGTGGTTCTTCACTATTTTCC SRP19-

Figure S2 .

 S2 Figure S2. Cellular localization analysis of SP20H and F118A. (A) Co-localization of B7-H3 and SP20H, F118A in Hela cells were visualized by confocal immunofluorescent imaging. Red: Alexa Fluor 594-labeled B7-H3 staining; Green: GFP fusion proteins, N'/C': GFP fused to the N or C terminus of SP20H; Blue: DAPI indicates nuclei. 


Figure S3 .

 S3 Figure S3. Treatment with LPS and H2O2 have no significant effect on B7-H3 expression in cancer cells. (A-B) Histograms show the surface B7-H3 expression on DU145 cells after 16hr-treatment with different concentrations of LPS (0, 25, 50, 100, 


Figure S4 .

 S4 Figure S4. Effect of SP20H depletion on tumor surface marker expression and tumor growth. (A) Representation immunohistochemical stains of indicated markers on tumors generated after subcutaneous injection of control, CD276-, or SP20Hknockout SK-OV-3 cells in NOD-SCID mice (n=5). Brown: indicated antigens; Blue: Mayer hematoxylin nuclear counterstain. Scar bars are 50μm. (B) Pictures of tumors derived from control and Sp20h-KO 4T1 cells. (C) Weight of the harvested 4T1 tumors after 25 days growth (n=5; two-tailed Student's t-test; ****P <0.0001; ns, no significant). 


BMJFigure S5 .

 S5 Figure S5. Representative gating strategies for flowcytometry analysis. (A) Gating strategy for figure 5D. (B) Gating strategy for figure 5F. 


BMJFigure S6 .

 S6 Figure S6. In vitro killing assay of αB7-H3-DM1 against SK-OV-3 cells with different expression of B7-H3. (A) Representation images of different concentration of αB7-H3-DM1 treated or untreated SK-OV-3-GFP cells from each group were shown. 




  


Table S1 . Sequence information of primers and sgRNAs sgRNA 20nt targeting sequences

 S1 
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	Supplemental material	placed on this supplemental material which has been supplied by the author(s)	J Immunother Cancer J Immunother Cancer
	shEIF4E-F		aattCATATCCAGTTGTCTAGTACTCGAGTACTAGACAA CTGGATATGTTTTTTTat
	sgCD276 sgSP20H-1 shEIF4E-R		CACAGGGCAACGCATCCCTG TGATAACCACAAATGGACCC AAAAAAACATATCCAGTTGTCTAGTACTCGAGTACTA GACAACTGGATATG
	sgSP20H-2 Sramble shRNA-	GGATATTCTCTGATGCTCAG aattCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGC
	sgF118A F		CATCTTCCGGATCAGATCAT GACTTAACCTTAGGTTTTTTTat	
	sgEIF4E-1 Sramble shRNA-sgEIF4E-2 R	AAAAAAACCTAAGGTTAAGTCGCCCTCGCTCGAGCG AAACTTGGCAAGCAAACCTG AAACTTGGCAAGCAAACCTG AGGGCGACTTAACCTTAGG
	sgMNK1-1 Genome PCR		TCGGAGTAGGGTGTTTCGAG Sequences (5'-3')	
	sgMNK1-2		CCATCGCAGATGGTGACAGG	
	sgSRP72		GCAGCTTCAGGGTCGAACAG	
	sgSRP9		GATGCCGCAGTACCAGACCT	
	sgSRPR		AGCATGTCTACACGACGCTG	
	sgSRP14		TATAGACGCTGCCCGACGTC	
	sgGTF3C4		GGATAGGGTGTTCAACCCTG	
	sgSRP19		CCAATACAGAGGCAGAGTCC	
	sgGOLGA6C		TTGTAGCTGTTCCACCACAG	
	sgSp20h-1		TGACAACCATAAGTGGACCC	
	sgSp20h-2		TTCTTCATAAGGCAATCGGA	
	shRNA		Sequences		
	shSP20H-F1		aattGCTTGTTATGCAAGAGACTCTCGAGAGTCTCTTGC ATAACAAGCTTTTTTTat
	shSP20H-R1		AAAAAAAGCTTGTTATGCAAGAGACTCTCGAGAGTC TCTTGCATAACAAGC
	shSP20H-F2		aattGCAACAAGCTTTAGAACTACTCGAGTAGTTCTAAA GCTTGTTGCTTTTTTTTat
	shSP20H-R2		AAAAAAAAGCAACAAGCTTTAGAACTACTCGAGTAG TTCTAAAGCTTGTTGC
	shCD276-F1		aattTTCAGCCTGGCACAGCTCAACCTCATCTGCTCGAG CAGATGAGGTTGAGCTGTGCCAGGCTGAATTTTTTTat
	shCD276-R1		AAAAAAATTCAGCCTGGCACAGCTCAACCTCATCTGC TCGAGCAGATGAGGTTGAGCTGTGCCAGGCTGAA
	shCD276-F2		aattTCGTGTGCTGGAGAAAGATCAAACAGAGCCTCGA GGCTCTGTTTGATCTTTCTCCAGCACACGATTTTTTTat
	shCD276-R2		AAAAAAATCGTGTGCTGGAGAAAGATCAAACAGAGC CTCGAGGCTCTGTTTGATCTTTCTCCAGCACACGA
			, et al. Zhao S , et al. Zhao S	J Immunother Cancer J Immunother Cancer	2022; 2022;	:e004875. 10 :e004875. 10	doi: 10.1136/jitc-2022-004875 doi: 10.1136/jitc-2022-004875



  R CTCTCCATGAGCAGTAGTCCBMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) 


	Supplemental material	placed on this supplemental material which has been supplied by the author(s)	J Immunother Cancer J Immunother Cancer
	GOLGA6C-F	CTGTGCATCAAGAGGAGG		
	GOLGA6C-R	CTCCTGCTCCTGGAGTC		
	QPCR	Primer sequences			
	qhCD276-F	CCTGAGTCCCAGAGTCGG		
	qhCD276-R	CAGGGCTCCTGTGAGG		
	qhSP20H-F1	GACTTGTATATTGAAG		
	qhSP20H-R1	TGGTCTCGGAATCTGATCC		
	qhSP20H-F2	AGCGTACATAAGTTATCTC		
	qhSP20H-R2	ACAGATTTTCTTCCACTTG		
	qhβ actin-F	CCTTCCTGGGCATGGAGTC		
	qhβ actin-R	TGATCTTCATTGTGCTGGGTG		
		, et al. Zhao S , et al. Zhao S	J Immunother Cancer J Immunother Cancer	2022; 2022;	:e004875. 10 :e004875. 10	doi: 10.1136/jitc-2022-004875 doi: 10.1136/jitc-2022-004875
Shasha Zhao, Yuelong Wang, Nian Yang, Min Mu, Zhiguo Wu, Hexian Li, Xin Tang, Kunhong Zhong, Zongliang Zhang, Cheng Huang, Ting Cao, Meijun Zheng, Guoqing Wang, Chunlai Nie, Hui Yang, Gang Guo, Liangxue Zhou, Xi Zheng, Aiping Tong
Genome-scale CRISPR–Cas9 screen reveals novel regulators of B7-H3 in tumor cells