S100A16 regulated by Snail promotes the chemoresistance of nonmuscle invasive bladder cancer through the AKT/Bcl-2 pathway

Figures & data

Table 1 The reaction conditions of RT-PCR

	Temperature (°C)	Time (s)	Cycle no.
Denaturation	95	15	30
Annealing/elongation	60	60	30
Dissolution curve analysis	95	15	1
	60	60	1
	95	15	1
	60	15	1

Table 2 The differential expression of S100 family in M-RT4 and RT4 cells

Accession	Gene Symbol	Gene ID	Coverage	Score	PSMs	Peptides	Unique Peptides	AAs	MW [kDa]	calc. pI	M-RT4	RT4	M-RT4-vs-RT4.FC	Regulation
NP_001303936.1	S100A16	140576	38.83	27.36927	7	6	6	103	11.79397	6.78565	1.26431	0.77363	1.63426	Up
NP_001019381.1	S100A13	6284	38.78	23.05108	7	4	4	98	11.46409	6.16357	1.30831	0.73574	1.77823	Up
NP_005969.1	S100A2	6273	40.21	119.30102	45	11	11	97	10.97832	4.77979	0.19467	1.6941	0.11491	Down
NP_005971.1	S100P	6286	38.95	78.21053	22	7	7	95	10.39322	4.88135	0.33734	1.5718	0.21462	Down
NP_005611.1	S100A11	6282	36.19	67.59417	24	6	6	105	11.73283	7.12256	0.50537	1.42679	0.35419	Down
NP_002952.1	S100A4	6275	18.81	9.6181	3	2	2	101	11.72073	6.11279	0.40466	1.51419	0.26725	Down
NP_055439.1	S100A6	6277	46.67	92.00726	43	10	10	90	10.17334	5.47803	0.85001	1.13051	0.75188	No change
NP_002957.1	S100A10	6281	34.02	16.6936	6	4	4	97	11.19551	7.37158	1.17307	0.85224	1.37645	No change

Note: Upregulation refers to an FC>3/2, while downregulation refers to an FC<2/3.

Abbreviations: FC, fold change.

Figure 1 S100A16 expression was upregulated in M-RT4 cells. (A) RT-PCR results showed the mRNA expression level of S100A16 in M-RT4 and RT4 cells. The results are presented as 2^-ΔΔCt values (ΔCt=Ct(S100A16) – Ct(GAPDH). ΔΔCt=ΔCt(M-RT4) – ΔCt(RT4)). (B) Western blot results showed the protein expression level of S100A16 in M-RT4 and RT4 cells. (C) The expression site of S100A16 in cells indicated by immunofluorescence (20× magnification). The scale bar represents 25 μm. *p<0.05; **p<0.01.

Figure 2 Knockdown of S100A16 could restore the sensitivity of M-RT4 cells to MMC. (A) The success of the knockdown assay was confirmed by Western blot. Control: transfection reagent and transfection medium (without siRNA). S1: 20 pmol siRNA. S2: 40 pmol siRNA. S3: 60 pmol siRNA. S4: 80 pmol siRNA. (B) Cell viability after MMC addition analyzed by a CCK8 assay. The concentration of MMC ranged from 0.78 to 100 μg/mL. si-S100A16: 60 pmol siRNA. (C) The IC50 of MMC for M-RT4 and RT4 cells. IC50: half maximal inhibitory concentration. MMC: mitomycin C. *p<0.05.

Figure 3 Knockdown of S100A16 could suppress the AKT/Bcl-2 pathway to promote apoptosis in M-RT4 cells. (A) The effect of S100A16 knockdown on EMT-related molecules in M-RT4 cells. Control: transfection reagent and transfection medium (without siRNA). si-S100A16: 60 pmol siRNA. (B) The effect of Snail knockdown on S100A16 expression. Control: transfection reagent and transfection medium (without siRNA). S1: 20 pmol Snail siRNA. S2: 40 pmol Snail siRNA. S3: 60 pmol Snail siRNA. (C) The effect of S100A16 knockdown on apoptosis-related molecules in M-RT4 cells. Control: transfection reagent and transfection medium (without siRNA). S1: 20 pmol S100A16 siRNA. S2: 40 pmol S100A16 siRNA. S3: 60 pmol S100A16 siRNA. S4: 80 pmol S100A16 siRNA. pAKT: phospho-AKT (S473). (D) Apoptosis of M-RT4 cells after S100A16 knockdown. Q4: early apoptosis. Q2: late apoptosis. Q2+Q4: total apoptosis. Control: transfection reagent and transfection medium (without siRNA). si-S100A16: 60 pmol siRNA. *p<0.05; **p<0.01.