Fig 1: Expression levels of selected transcripts in micro-dissected non-injured hemalaun-stained glomerular substructures. Nine cases with acute tubular injury and mild interstitial inflammation, but without any glomerular disease were used (cohort I). WT1 (A), GLEPP1 (B) and PAX2 (C) seem to be reliable discriminators for proper micro-dissection of glomerular tufts vs. PECs. For comparison, periglomerular tubulointerstitium is shown. There were significant differences between glomerular tufts and PECs regarding all markers. Relative expression was calculated using the geometric mean of PGK1 and GAPDH as normalization factor. There was no expression of WT1 and GLEPP1 (beside one outlier) in periglomerular tubulointerstitium. In one patient case, no amplification of target and reference transcripts was detectable in PECs. One outlier was excluded in WT1 and GLEPP1 transcript expression in glomerular tufts. p-values were calculated by the Wilcoxon matched-pairs signed rank test (*p < 0.05; **p < 0.01). For comparison, immunostaining of WT1 (D), GLEPP1 (E) and PAX2 (F) is given in one selected case. WT1 (D) and GLEPP1 (E) are strongly expressed in podocytes and to a lower extend also in PECs, but not in the tubulointerstitium. PAX2 is expressed in PECs and tubular epithelial cells (F). Magnification ×400 or ×200
Fig 2: PTPRO deficiency reduces lapatinib sensitivity in Ptpro −/− and littermate-control MEFs stably transfected with Erbb2 and ERBB2-positive breast cancer cells. (A) Erbb2/Ptpro +/+ MEFs and Erbb2/Ptpro −/− MEFs were plated in 96-well plates and treated with 10-fold serial dilutions of lapatinib ranging from 0.001 to 10 μM for 72 h. The percentage cell viability was evaluated by MTT assay. (B) Erbb2/Ptpro +/+ MEFs and Erbb2/Ptpro −/− MEFs were incubated with lapatinib (1 μM) for 2 weeks to allow colony formation (left panel) and the number of colonies was compared (right panel; **p < 0.01). (C) Erbb2/Ptpro +/+ MEFs and Erbb2/Ptpro −/− MEFs were incubated with lapatinib ranging from 0.001 to 10 μM for 72 h. Cell death was determined using a Cell Death detection ELISA and absorbance was measured at 405 nm. (D) RT-qPCR of PTPRO in SKBR3 and BT474 cells with PTPRO depleted. (E) The protein level of PTPRO in SKBR3 and BT474 cells with PTPRO knockdown were analyzed by western blot. (F) Cell inhibition ratio analysis of SKBR3 and BT474 cells with PTPRO depleted was evaluated by MTT assay, 72 h after treatment with 10-fold serial dilutions of lapatinib ranging from 0.001 to 10 μM. Data were shown as the means of three independent experiments or representative data. Error bars indicate SEM; n.s., not statistically significant; *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test or a one-way ANOVA with post hoc intergroup comparisons, where appropriate.
Fig 3: PTPRO is downregulated in lapatinib-resistant ERBB2-positive breast cancer cells. (A) The mRNA level of PTPRO in the lapatinib resistant ERBB2-positive breast cancer cells, SKBR3-lapR and BT474-lapR, compared with their parental cells, SKBR3-P and BT474-P. The dataset GSE38376 and GSE16179 were get from GEO (https://www.ncbi.nlm.nih.gov/geo/). (B) Inhibition ratio analysis of SKBR3-lapR and SKBR3-P (upper panel), BT474-lapR and BT474-P (bottom panel) was evaluated by MTT assay after treatment with lapatinib. (C) Colony formation abilities of SKBR3-lapR and SKBR3-P (left panel), BT474-lapR and BT474-P (right panel) were evaluated after treatment with lapatinib, and the number of colonies was counted (bottom panel). (D) The PTPRO protein expression derived from lapatinib resistant ERBB2-positive cells and their parental cells were assessed by western blot analysis. Data were shown as the means of three independent experiments or representative data. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test or one-way ANOVA with post hoc intergroup comparisons, where appropriate.
Fig 4: PTPRO sensitizes lapatinib in ERBB2-positive breast cancer cells. (A) The level of PTPRO mRNA in SKBR3-lapR and BT474-lapR cells with PTPRO overexpression were analyzed by RT-qPCR. (B) The protein level of PTPRO in SKBR3-lapR and BT474-lapR cells with PTPRO overexpression were analyzed by Western blot. (C) Cell inhibition ratio analysis of SKBR3-lapR and BT474-lapR cells with PTPRO overexpression was evaluated by MTT assay, 72 h after treatment with 10-fold serial dilutions of lapatinib ranging from 0.001 to 10 μM. Data were shown as the means of three independent experiments or representative data. Error bars indicate SEM. **p < 0.01, ***p < 0.001 by one-way ANOVA with post hoc intergroup comparisons, where appropriate.
Fig 5: Downregulation of PTPRO correlates with poor prognosis in ERBB2-positive breast cancer patients. (A) The mRNA level of PTPRO in cancer tissues and corresponding adjacent normal breast tissues was determined in 37 breast cancer patients. (B) Paired t-test revealed the significant alteration of PTPRO mRNA in tissue samples (***p < 0.001). (C) Representative images are shown as IHC scores (0–9); Normal kidney and breast tissues were positive references. (D) The immunoreactivity scores of PTPRO in normal breast tissues and breast cancer tissues were compared using the Mann-Whitney U test (***p < 0.001). (E) Images of IHC staining of PTPRO and Ki67 in serial sections of two representative human breast cancer specimens (left panel); Pearson’s correlation analysis of Ki67 index and PTPRO score in 180 human breast cancer specimens (right panel). (F) Relapse-free survival analysis in breast cancer patients according to PTPRO high and low expression levels (log-rank test). These data came from the publicly available cohorts (GSE3494, GSE7390, GSE6532, GSE1456, GSE2034; the top tertile (359 cases) of ERBB2 mRNA expression was assumed to be ERBB2-positive tumors).
Supplier Page from MilliporeSigma for Anti-PTPRO antibody produced in rabbit