Fig 2: DRS transcriptome analysis of purified RNA from MDA-MB-231 protrusions. (A) Comparison of DRS and RT-qPCR analyses. RNA localization ratios were normalized to ARPC3 mRNA given the value 1. (B) Annotation analysis of the 100 most MDA-MB-231 protrusion localized mRNA compared to the total group of mRNAs expressed with = 5 tpm. Annotations were made using the IPA Ingenuity platform. (C) RT-qPCR confirmation analysis of selected MDA-MB-231 protrusion localized RNA identified by DRS. RNA localization ratios are normalized to ARPC3 mRNA. (D) Western blot analysis. Western blotting was performed on pooled material from three independent Boyden chamber experiments representing cell body fraction (CF) and protrusion fraction (PF). a-Tubulin Western blot analysis was used to equalize the loaded protein amounts. Subsequent western blot analyses were performed with antibodies for Histone H3, ZEB1, ANP32B and ACTB.

Fig 3: Epithelial–mesenchymal transition (EMT) accompanies HK2 upregulation and p-PDH downregulation. (a) Photomicrographs of the morphological change of SW480 cells. (b) Western blot assays of E-cadherin, Vimentin, HK2, p-PDH, and ACTB expression in pre-EMT and post-EMT cells. Samples of post-EMT cells were harvested at 72 h. (c) Hexokinase activity in pre-EMT and post-EMT cells. Samples of post-EMT cells were harvested at 72 h. (d) Pyruvate dehydrogenase activity in pre-EMT and post-EMT cells. Samples of post-EMT cells were harvested at 72 h. (e) Relative transcript (mRNA) levels of HK2, PDK1, and PDP2 after inducing EMT for 0 and 48 h. The values at 0 h have been normalized to 1, and the data are expressed as fold. *Statistically significant.

Fig 4: Respiratory complex II, but not complexes III and IV, is upregulated in XP-C cells. (A) OCR was measured using 4 × 106 cells in standard reaction medium. Cells were permeabilized with 30 µM digitonin, with the following sequential additions: 5 mM succinate, 0.25 µM CCCP, 1 µM AA and 200 µM TMPD + 2 mM ascorbate. The figure shows a representative trace OCR experiment. (B) The rates of oxygen consumption were calculated from data obtained as shown in panel A. (C) CII + CIII activity was measured using 12 µg of crude mitochondrial protein. Reduction of cyt c was monitored at 550 nm at 37 °C for 5 min. It was only considered the range where slopes of the reactions were linear. (D) Expression analysis of SDHA and SDHB genes by RT-qPCR. Fold-change in mRNA expression of unXP-C relative to the WT cell line. ACTB was set as reference gene for all genes analyzed. (E) Western blotting of CI subunit SDHB and ATPase subunit F1a. SDHB protein expression level was normalized against F1a in unXP-C relative to corrXP-C cells. The data represent mean ± SD of 3 independent experiments. *p < 0.05 and **p < 0.01.

Fig 5: Pianp is sorted into axons and dendrites in neurons and controls presynaptic GBR inhibition as well as gene expression in the brain. a Overexpressed PIANP is sorted into axons and dendrites in cultured hippocampal neurons. At 7 days in vitro, PIANP-mCherry and GFP plasmids were transfected in wild-type hippocampal neurons and overexpressed for 6 h before fixation. GFP served as a volume marker. Representative co-transfected cell, demonstrating axonal and somatodendritic localization of PIANP-mCherry. Higher magnification images of axonal and dendritic segments correspond to insets in overview image. Map2 was used as a marker of dendrites (arrow) and Ankyrin-G was used as a marker of the axon initial segment (arrowhead). Scale bars: 30 µm (overview images), 5 µm (high-magnification images). b–d Reduced presynaptic GBR-mediated inhibition in PianpKO mice. b Sample traces of evoked EPSCs from CA1 pyramidal neurons of PianpKO and control mice. EPSCs were evoked by train stimulation at 1 Hz or 40 Hz in the absence (black traces) and in the presence of 100 µM baclofen (red). The 1st EPSC in the presence of baclofen is scaled to that in the absence of baclofen (gray traces) to compare paired pulse ratios (PPR) of the 1st to the 10th EPSC. Scale bars, 50 ms and 50 pA. c Train stimulation at 40 Hz reveals a significant deficit in presynaptic GBR inhibition in PianpKO mice. Control (left: eight cells, three mice) and PianpKO (middle: nine cells, four mice) EPSCs are scaled to the 1st EPSC (Control 1 Hz, p = 0.1737; PianpKO 1 Hz, p = 0.3836; Control 40 Hz, p = 0.0022; PianpKO 40 Hz, p = 0.0602). Baclofen-mediated EPSC amplitude reduction is significantly decreased in PianpKO mice during 40 Hz train stimulation (right: 1 Hz, p = 0.0705; 40 Hz, p < 0.0001). d Impaired GBR-mediated inhibition of the mEPSC frequency in CA1 pyramidal neurons PianpKO mice (p = 0.003). The mEPSC amplitude remained unchanged (p = 0.7576). Basal mEPSC frequency (p = 0.3304) and amplitude (p = 0.9653) were unaltered. Data are from four cells from two control mice and seven cells from three PianpKO mice. e The heat map of significantly dysregulated genes identified by microarray transcriptome analysis of RNA isolated from amygdala, cortex, hippocampus, and hypothalamus of control and PianpKO mice (n = 4 for amygdala of control mice, n = 5 for each other group). QRT-PCR of amygdala, cortex, hippocampus, and hypothalamus of control and PianpKO mice for Avp, Erdr1, Pianp and Scn4b (n = 5 for each group). Bars indicate gene expression relative to control in log2 fold change (FC) normalized to Actb. f ISH for Erdr1 in the CNS. Erdr1 (in red) is broadly expressed in the cerebrum of control mice (upper panel) and its expression is reduced in the cerebrum of PianpKO mice (lower panel). Scale bars indicate 1 mm. Images are representative for n = 3. Bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, c two-way ANOVA, d, e two-tailed unpaired t-test