Fig 2: Expression of PLAC8 in lung cancer and its association with clinicopathological features in lung cancer TMAs.a PLAC8 mRNA (upper) and protein (lower) levels in seven LC cell lines were detected by Western blot and RT-PCR analysis. GAPDH was used as the loading control. b H1299 and H322 cells were stained for PLAC8 (green staining) and analyzed using fluorescence microscopy. Nuclei were stained with DAPI (blue staining). c Four representative images from the IHC analysis of PLAC8 expression (negative, weak, moderate, and strong) obtained from TMA LC samples. d The survival curve was compared using the Kaplan–Meier method according to the PLAC8 expression level. High PLAC8 was related to moderate and strong expression levels, while low PLAC8 was related to negative and weak expression levels. PLAC8 overexpression significantly predicted poor outcomes in LC patients (P = 0.0102). *P < 0.05

Fig 3: PLAC8 expression was increased in ADM resistance breast cancer cells. A, B. The protein and mRNA expression level of PLAC8 in various breast cancer cell lines. C Protein and mRNA expression of PLAC8 in MCF‐7 and MCF‐7/ADMR cells. D. Immunofluorescence staining for PLAC8 (red) and DAPI nuclear staining (blue) in MDA‐MB231, MCF‐7 and MCF‐7/ADMR cell lines. Scale bar = 25 µm. E. Altered PLAC8 expression in MCF‐7 and MCF‐7/ADMR cell upon ADM treatment (0.5, 1 and 2 µg/ml, 48 h). F. Colony formation indicated the proliferative potential of MCF‐7 and MCF‐7/ADMR cells with ADM treatment (5 µg/ml). G. The sensitivity of MCF‐7, MDA‐MB231, T47D and MCF‐7/ADMR cells to ADM. Each bar represents the mean ± SD of three independent experiments. *P < .05, **P < .01, ***P < .001

Fig 4: Autophagy activity after pharmacological inhibition of autophagy in breast cancer cells with PLAC8 overexpression or silencing A, B. MCF‐7 and MCF‐7/ADMR cells were treated with bafilomycin A1 (Baf‐A1) or DMSO, both in normal conditions and with ADR treatment, and autophagosome formation were measured by GFP‐LC3 puncta counting. Quantification of representative pictures showing the functionality of the sensor. Manual counting of red and yellow dots was performed. Autophagic flux is estimated as the ratio between red and yellow dots. C. The expression of phosphor‐S6, LC3, PLAC8 and p62 in MCF‐7 and MCF‐7/ADMR cells treated with Baf‐A1 or DMSO, both in normal conditions and with ADR treatment. GAPDH shown as a loading control. D‐G. MCF‐7/CON, MCF‐7/C1 and MCF‐7/C2 cells were treated with bafilomycin A1 (Baf‐A1) or DMSO, both in normal conditions and with ADR treatment, and autophagosome formation were measured by GFP‐LC3 puncta counting. Quantification of representative pictures showing the functionality of the sensor. Manual counting of red and yellow dots was performed. Autophagic flux is estimated as the ratio between red and yellow dots. H. The expression of phosphor‐S6, LC3, PLAC8 and p62 in MCF‐7/CON, MCF‐7/C1 and MCF‐7/C2 cells treated with Baf‐A1 or DMSO, both in normal conditions and with ADR treatment. GAPDH shown as a loading control. I‐L. ADMR/NC, ADMR/siPLAC8‐1 and ADMR/siPLAC8‐2 cells were treated with bafilomycin A1 (Baf‐A1) or DMSO, both in normal conditions and with ADR treatment, and autophagosome formation were measured by GFP‐LC3 puncta counting. Quantification of representative pictures showing the functionality of the sensor. Manual counting of red and yellow dots was performed. Autophagic flux is estimated as the ratio between red and yellow dots. M. The expression of phosphor‐S6, LC3, PLAC8 and p62 in ADMR/NC, ADMR/siPLAC8‐1, ADMR/siPLAC8‐2 and ADMR/siPLAC8‐3 cells treated with Baf‐A1 or DMSO, both in normal conditions and with ADR treatment. GAPDH shown as a loading control

Fig 5: PLAC8 enhances tumour growth in vitro and in vivo by decreasing apoptosis. A, Tumour volume (measured twice a week) and tumour weight (day 18) were compared between the PLAC8‐overexpressing and vector groups, which showed tumour volumes and weights that were comparable to those observed in the vector group. The tumour volume was calculated using the following equation: V = (width2 × length)/2. The error bars correspond to the means ± SD. B, IHC staining showed the expression of PLAC8, Ki67 and cleaved caspase‐3. 20× and 40×; the scale bar represents 100 and 50 μm, respectively. C, Assessment of apoptosis by TUNEL assays of tumours from mice in the PLAC8 overexpression and vector groups on day 18. The scale bar represents 20 μm. An enlarged view of the boxed area from each group confirms the presence of TUNEL‐positive cells. The values are presented as the means ± SD * P < 0.05; ** P < 0.01; NS, not significant