Fig 2: PD-1positive and PD-1negative CD8+ T cells within MC38-derived tumor microenvironment show different mitochondrial morphologies. (A) Draining lymph node (DLN)-derived T cells and tumor-infiltrating lymphocytes (TILs) have been isolated from 18-day-old MC38-derived tumor mass grown in WT c57BL/6 mice, and the expression of the indicated mitochondrial-shaping proteins has been evaluated into PD-1negative (PD-1neg) and PD-1positive (PD-1pos) CD8+ T-cell subpopulations. Representative gating strategy to distinguish PD-1neg and PD-1pos CD8+ T cells is shown on the left. Graphs on the right indicate the normalized median fluorescence intensity (MFI) of the indicated proteins evaluated by intracellular flow cytometry in PD-1neg and PD-1pos CD8+ T cells from the same mice and expressed as normalized intensity ratio relative to secondary antibodies alone (pS616-Drp1 n = 14; Drp1 n = 20; Mfn1 and Fis1 n = 9; Mfn2 and Mff n = 7; Opa-1 and pS637-Drp1 n = 6; paired t-tests). (B-C) PD-1negative (PD-1neg) and PD-1positive (PD-1pos) CD44+ CD45+ CD8+ T cells have been sorted and purified form 18-day-old MC38-derived tumor mass grown in WT c57BL/6 mice. Gating strategy is shown in Fig. S1. Mitochondrial morphology was evaluated by immunofluorescence (anti-TOM20 staining) and upon z-stack reconstruction. In (B), cells have been fixed immediately after purification and processed for immunostaining. In (C), cells have been stimulated for 2 h in the presence of beads coated with aCD3/28 Abs plus PD-L1 and then fixed and processed for immunostaining. For each panel, representative images of the observed mitochondrial morphologies are shown on the left, while graphs on the right show the distribution of cells into the indicated category according to mitochondrial morphology in PD-1neg and PD-1pos CD8+ T cells (n = 230 cells each condition from 8 [B, unstimulated] or 10 [C, stimulated with beads] pooled mice; chi-square tests). (D) Representative microphotographs of double-marker immunohistochemistry for PD-1 (rose; rose arrows) and Drp1-pSer616 (brown; yellow arrows) expression in lymphoid elements infiltrating human colon cancer. The graph on the right indicates the percentage of pDrp1pos cells among all PD-1neg and PD-1pos CD8+ T cells (n = 7 patients, see Fig. S1G for the triple immunostaining). Data are shown as mean ± SEM. Scale bar: 5 µm in B and C and 50 µm in D. Significance is indicated as follows: *= P < 0.05; ***= P < 0.001. Statistical tests used: paired t-test (A); chi-square test (B-C); unpaired t-test (D).

Fig 3: Mechanical force-led alteration of oxidation pattern in PDLSCs was induced by mitophagy. A The protein level of MFF, p-MFF, PINK1, p-PINK1, PARKIN, and p-PARKIN in PDLSCs after stretching for 0, 6, 12, and 24 h. B The morphology of mitochondria in CsA pretreated PDLSCs after 0 h and 24 h of stretching visualized by TEM. Red arrow: mitochondria. Scale bar = 1 μm. C The MMP in CsA pretreated PDLSCs after stretching for 0 h and 24 h visualized by TMRM staining. Scale bar = 100 μm. D The relative RNA expression of LDHA and COX4 in CsA pretreated PDLSCs after being stretched for 0 h and 24 h. E The protein level of LDH and COX4 in CsA pretreated PDLSCs after being stretched for 0 h and 24 h. F The level of lactic acid in CsA pretreated PDLSCs after stretching for 0 h and 24 h. G The level of ROS in CsA pretreated PDLSCs after stretching for 0 h and 24 h. Scale bar = 100 μm. *P < 0.05. ns no significance

Fig 4: PRKCQ-AS1 regulates glycolysis and mitochondrial dysfunction through IGF2BPs/PRMT7 signaling pathway in PTC cells.A–F TPC-1 cells were transfected with vector, PRKCQ-AS1, PRKCQ-AS1+shScramble, or PRKCQ-AS1+shPRMT7; K1 cells were transfected with vector, PRKCQ-AS1, PRKCQ-AS1 +shScramble, or PRKCQ-AS1+shPRMT7. A–D Glucose uptake, lactate production, ECAR and OCR were examined. E, F Glycolytic related proteins (PDK1, LDHA, HK2, GLUT1 and GPI) were determined by western blot (upper: TPC-1; lower: K1). G, H Mitochondrial ROS and ATP production were observed. I–J JC-1 staining was used to observe mitochondrial membrane potential. Scale bars, 50 μm. K The proportion of cells with elongated, intermediate and fragmented mitochondrial was quantified. Scale bars, 10 μm. L Western blot and qRT-PCR analysis for protein and mRNA expression levels of MFN1, MFN2, OPA1, DRP1, FIS1, and MFF (Left: TPC-1; Right: K1). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Fasting impairs glycolysis and reduces mitochondrial dysfunction of PTC cell lines in vitro and in vivo.A, B Fasting inhibited TPC-1 and K1 cell proliferation as measured by CCK8 and colony formation assay. C Fasting reduced glucose uptake and lactate production via glycolysis in TPC-1 and K1 cells. D, E ECAR, an indicator of glycolysis, was reduced, while OCR, which reflects mitochondrial respiration, was increased in TPC-1 and K1 cells cultured in the fasting mimic medium. F Fasting reduced the expression of rate-limiting glycolytic enzymes in glucose metabolism (PDK1, LDHA, HK2, GLUT1 and GPI) in TPC-1 and K1 cells, which were measured by western blotting. G Mitochondrial ROS and ATP production were measured in TPC-1 and K1 cells cultured in the fasting mimic medium. H JC-1 staining was used to observe mitochondrial membrane potential in TPC-1 and K1 cells cultured in the fasting mimic medium. Scale bars, 50 µm. I The proportion of cells with elongated, intermediate, and fragmented mitochondrial was quantified in TPC-1 cells cultured in the fasting mimic medium. Scale bars, 10 µm. J Western blot analysis for protein expression levels of MFN1, MFN2, OPA1, DRP1, FIS1, and MFF in TPC-1 and K1 cells cultured in the fasting mimic medium. K TPC-1 and K1 cells were injected into BALB/c mice. When the tumors were palpable, the mice were randomly assigned to the control group or the fasting mimic diet (FMD) group. Photograph of dissected tumors (n = 6) were shown. L The tumor volumes were measured every week. The FMD attenuated tumor growth in mice (n = 6), tumor weights, and tumor volumes by 4 weeks of cell transplantation. *P < 0.05, **P < 0.01, and ***P < 0.001.