Fig 1: PPM1D and SPOP attenuated the inhibition of cell proliferation induced by APPBP2 knockdown in NSCLC cells. (a) Ectopic expression of PPM1D or SPOP promoted colon formation on APPBP2 silenced A549 cells. Control was set as a blank group without protein overexpression. (b) The statistical results of colony formation indicate the impact of PPM1D and SPOP on NSCLC stable cells. A one-way ANOVA revealed a significant effect of group (F[4,10] = 324.5, p < 0.001 in A549 and F[4,10] = 63.45, p < 0.001 in H1299). (c) The proliferation effects of PPM1D or SPOP expression on A549 stable cells was measured by MTT assay. A one-way ANOVA revealed a significant effect of group (F[6,14] = 106.2, p < 0.001). (d) The proliferation effects of PPM1D or SPOP expression on H1299 stable cells was measured by MTT assay. A one-way ANOVA revealed a significant effect of group (F[6,14] = 393.9, p < .001). (e) PPM1D and SPOP had a significant impact on cell apoptosis of A549 stable cells by Annexin V and flow cytometric analysis. A one-way ANOVA revealed a significant effect of group (F[6,14] = 338.4, p < 0.001). (f) PPM1D and SPOP had a significant impact on cell apoptosis of H1299 stable cells by Annexin V and flow cytometric analysis. A one-way ANOVA revealed a significant effect of group (F[6,14] = 310.9, p < 0.001). The addition or non-addition of plasmid or virus is present as “+” and”-”, respectively. Data are presented as mean ± SEM. The difference between means was compared by Tukey's Multiple Comparison test. 0.01 < *p < 0.05 and ***p < 0.001 for indicated comparison. n.s. refers as not significant.
Fig 2: PPM1D and SPOP attenuated the inhibition of cell migration and invasiveness induced by APPBP2 knockdown in NSCLC cells. (a) Effects of PPM1D and SPOP on cell migration and invasion of A549 stable cells by transwell, n = 3. (b) Effects of PPM1D and SPOP on cell migration and invasion of H1299 stable cells by transwell, n = 3. (c) The statistical results of PPM1D and SPOP on cell migration and invasiveness of A549 stable cells by transwell. A one-way ANOVA revealed a significant effect of group (F[6,14] = 35.7, p < 0.001). (d) The statistical results of PPM1D and SPOP on cell migration and invasiveness of H1299 stable cells by transwell. A one-way ANOVA revealed a significant effect of group (F[6,14] = 72.58, p < 0.001). The addition or non-addition of plasmid or virus is present as “+” and “-”, respectively. Data are presented as mean ± SEM. The difference between means was compared by Tukey's Multiple Comparison test. 0.001 < **p < 0.01 and ***p < 0.001 for indicated comparison. n.s. refers as not significant. Scale bars: 50 µm.
Fig 3: APPBP2 regulated the expression of PPM1D and SPOP in NSCLC tissues and cells. (a) The heatmap of gene expression profiling organized by APPBP2 expression level by reanalysing the TCGA data. Samples were derived from human LUAD patients. Red circles indicate the genes to be studied. (b,c) PPM1D mRNA level (b) and SPOP mRNA level (c) had a positive linear relationship with the expression of APPBP2. (d) Immunostaining of PPM1D (brown colour) of human LUAD tissues and adjacent normal tissues. (e) Immunostaining of SPOP (brown colour) of human LUAD tissues and adjacent normal tissues. DAPI in (d) and (e) is indicated as blue colour. (f) PPM1D mRNA and SPOP mRNA expression level of human LUAD tissues and adjacent normal tissues were measured by RT-PCR. Yellow colour and cyan colour represent normal and tumour samples, respectively. n = 3. (g) RT-PCR results showed PPM1D mRNA and SPOP mRNA expression level in APPBP2 silenced cells of A549 or H1299 (n = 3). (h) PPM1D and SPOP were examined by western blotting on APPBP2 silenced A549 and H1299 stable cells. GAPDH acts as the internal standard. (i) co-IP between the APPBP2-flag and PPM1D or SPOP on A549 or H1299 cell contexts. (j) co-IP between PPM1D-His and APPBP2 or SPOP on A549 or H1299 cell contexts. Data are presented as mean ± SEM. (Two-tailed t-test for group comparison) 0.01 < *p < 0.05, 0.001 < **p < 0.01, ***p < 0.001 for indicated comparison. Scale bars: 50 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 4: Graphical signalling pathway of the effect of APPBP2 on NSCLC via PPM1D and SPOP.The blue lines indicate the activation of the targets according to our experiments or published researches. The red lines indicate the possible inhibition of targets according to our reasonable speculation. AR: androgen receptor. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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