Fig 2: The relocation of PHB to one cellular end is associated with microtubule transportation and may have originated from mitochondrial release(A) Immunostaining for PHB and ?-tubulin in CRC cell lines and tissues, where the white arrow indicates ?-tubulin. Scale bar: 5 µm and 10 µm. (B) PHB and kinesin (a motor for transportation) were co-localized in CRC cells. Scale bars: 5 µm. (C) PHB and microtubules were co-immunostained in CRC cell sublines that differed in metastatic potential. White arrows indicate the directionality of PHB and microtubules. Scale bar: 10 µm. (D) PHB and microtubule staining of cells without (Col-0 min) or with colchicine treatment (Col-60 min), and then colchicine was replaced by RPMI-1640 (RE·RPMI-1640). Scale bar, 10 µm, col = colchicine. (E) Wound-healing assays of CRC cells in which PHB relocated to the direction of the wound. Polarity assays of CRC cells in which PHB was relocated to the direction of VEGF stimulation. Data are shown as means ± SEM. *P < 0.001. (F) PHB/COXIV and PHB/Miro-1 co-expressed in CRC cells. Scale bar: 10 µm. (G) Scatter plot graph showed that mitochondria were concentrated on one end of the CRC cells following VEGF stimulation. When Miro-1 was depleted, the concentrated distribution of mitochondria was impaired (VEGF(+)/siMiro-1). Data are shown as means ± SD, *P < 0.001. (H) Western blot analysis of control CRC cells (si-Control) and Miro-1-silenced cells (siMiro-1). (I) Quantitative analysis of wound-healing assays performed by calculating the percentage of cells within 200 µm of the wound; PHB was relocated in the direction of the wound after VEGF stimulation. Quantitative analysis of polarity assays was performed by calculating the percentage of cells, in which PHB relocation faced the direction of VEGF stimulation. Data are shown as means ± SEM. *P < 0.001. (J) Whole-cell lysates and mitochondrial fractions were isolated and probed for PHB.

Fig 3: SCFA supplementation mitigates neuroinflammation and promotes neuronal survival in the hippocampus post BCCAO. A, B Representative immunofluorescence images showing Iba-1+ cells in the hippocampus at day 14 post BCCAO (A) and the quantification analysis (B) (n = 6/group). Scale bar, 50 µm. C, D mRNA levels of inflammatory cytokines (IL-4, IL-10, TNF-a, IL-1ß, and IL-6) in the hippocampus at day 14 post BCCAO (n = 5/group). E Protein levels of CD16 and NF-?B and their quantification in the hippocampus at day 14 post BCCAO (band intensity normalized to ß-actin) (n = 6/group). F, G Representative immunofluorescence images (F) and mean fluorescence intensity (G) of hippocampal neurons in CA1, CA2, CA3, and DG in the different groups. (n = 6/group). Scale bar, 50 µm. H–K Western blot analysis for protein levels of caspase 3, cleaved caspase 3, Bcl2, Bax, Bcl-XL, Erk, and pErk and their quantification (band intensity normalized to ß-actin or COXIV) in the hippocampus (n = 6/group). The data represent the mean ± SEM. p < 0.05 was set as the threshold for significance. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to S+NaCl group. &&p < 0.01 compared to the S+SCFA group. +p < 0.05 compared to the B+NaCl 14d group. #p < 0.05, ##p < 0.01 compared to the B+NaCl 37d group

Fig 4: PINCH-1 level is increased in lung adenocarcinoma.a, b Human lung adenocarcinoma tissues and adjacent normal tissues from a microarray were stained with anti-PINCH-1 (P1) antibody (a). The samples in columns 1, 3, 5, 7, 9, 11, 13, 15, and 17 on rows a–i and columns 1, 3, 5, 7, 9, 11, and 13–18 on row j were derived from lung adenocarcinoma tissues. All other samples were derived from adjacent normal tissues. Bar = 1 mm. The mean intensities (arbitrary units) of PINCH-1 staining from lung adenocarcinoma tissues (n = 93) and normal adjacent tissues (n = 87) were quantified (b). c Higher magnification images of representative IHC staining shown in panel a obtained with anti-PINCH-1 antibody (brown) and haematoxylin counterstain (blue). Bar = 20µm. The boxed areas in the IHC staining were enlarged and shown in the upper right corner. d Kaplan–Meier survival curve of 90 lung adenocarcinoma patients. Patients were divided into two groups according to the mean intensities of PINCH-1 staining in cancer tissues of the tissue array (high expression: n = 57, low expression: n = 35, Log-rank (Mantel–Cox) test was used for the statistical analysis). e Lung adenocarcinoma was induced by administration of Ad-Cre into the lung of KrasLSL-G12D/+ mice. Sixteen weeks later, sections from the KrasLSL-G12D/+ mice administrated with Ad-Cre or without Ad-Cre as a control were analyzed by immunostaining with anti-PINCH-1 antibody. Bar = 20 µm. The boxed areas in the IHC staining were enlarged and shown in the upper right corner. The mean intensities of PINCH-1 staining in lung adenocarcinoma were quantified and compared to those of normal lung tissue (normalized to 1; normal group n = 36 fields from 6 mice, cancer group n = 49 fields from 6 mice; P < 0.0001) (e, right panel). Data in e represent mean ± SEM. Statistical significance was calculated using two-tailed unpaired Student’s t-test, ***P < 0.001. Source data are provided as a Source Data file.

Fig 5: Western blot analysis of mitochondrial proteins. Twenty micrograms of total cellular proteins from various cell lines was electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for (A) 8 mtDNA-encoding (ND1, ND3, ND4, ND4L, ND5, CYTB, CO2 and ATP8) and (B) 10 nucleus-encoding (NDUFA1, NDUFS1, NDUFA10, SDHB, UQCRC2, UQCRFS1, COXIV, COX17, ATP5B and ATP5F1) subunits of OXPHOS (CI, CII, CIII, CIV and CV represented complexes I, II, III, IV and V, respetively), TOM20 or GAPDH as a loading control, respectively. (C) Quantification of mitochondrial protein levels. Average relative ND1, ND3, ND4, ND4L, ND5, CYTB, CO2, and ATP8 content per cell, normalized to the average content per cell of TOM20 in TRUB1KO and WT cell lines. The values for the TRUB1KO cell lines are expressed as percentages of the values for the WT cell line. The error bars indicate two standard errors of the means, the horizontal dashed lines represent the average value for each group. The calculations were based on three independent determinations in each cell line. P indicates the significance, according to the t-test, of the differences between TRUB1KO and WT cell lines.