Fig 1: Circ_0013359 silencing reduces melanoma cell progression and is restored by anti-miR-136-5p treatment. A875 and SK-MEL-1 cells were transduced with si-NC, si-circ_0013359, si-circ_0013359 + anti-miR-NC, or si-circ_0013359 + anti-miR-136-5p, respectively. MiR-136-5p level (a and b), cell viability (c and d), colony numbers (e), apoptosis rate (f), cell cycle distribution (g and h), and the number of cell migration and invasion (i and j) were evaluated using appropriate methods. Glycolysis was assessed by measuring glucose consumption (k), lactate production (l), ECAR (m and n), and the protein levels of HK2 and LDHA (o and p). *P < 0.05.
Fig 2: Knockout circ_0001721 inhibited glycolysis in OS cells. HOS and U2OS cells were transfected with control, si-NC or si-circ_0001721. (A and B) QRT-PCR was used to detect the transfection efficiency. (C and D) QRT-PCR was used to detect glucose consumption in HOS and U2OS cells. (E and F) QRT-PCR was used to detect lactic acid production in HOS and U2OS cells. (G and H) QRT-PCR was used to detect the expression of HK2 in HOS and U2OS cells. *P<0.05.
Fig 3: Exosomal lncMMPA enhances macrophage-induced tumor progression. A Electron microscope observation of the morphology of TAMs-derived exosomes. Scale bar, 100 nm. B Western blot analysis of antigens (CD9, Alix and TSG101) in TAMs-derived exosomes (TAMs-exo). C The expression level of lncMMPA in the equal numbers of exosomes derived from MDMs (left) and TAMs (right) after treatment with RNase A alone or together with Triton X-100 was normalized to that of cel-miR-39-3p and shown as a ratio relative to the expression level in untreated exosomes derived from MDMs or TAMs. D Fluorescent staining was conducted in Hep3B cells following incubation with Dil-labeled (red) exosomes obtained from TAMs. Scale bar, 20 µm. E-H Co-culture of Hep3B cells with MDMs and pri-TAMs was done in Transwell systems for 6 d. Separately, Hep3B cells were treated with exosome from TAMs for 48 h. After these treatments, Hep3B cells were collected for the specified experiments. E Glucose consumption. F Lactate production. G ECAR. H Expression of GLUT1, HK2 and LDHA. I-L Hep3B cells were treated with exosomes from MDMs with or without overexpressing lncMMPA and then harvested for the specified experiments. I Glucose consumption. J Lactate production. K ECAR. L Expression of GLUT1, HK2 and LDHA. M, N Hep3B cells were injected orthotopically into the right flank of BALB/c nude mice. The mice were given intratumoral injection of the indicated exosomes from MDMs every 3 d. M Representative images of tumors in the xenografts. N Tumor growth curves. O-R Hep3B cells were treated with exosomes from TAMs with or without shlncMMPA transfection and then collected for the indicated experiments. O Glucose consumption. P Lactate production. Q ECAR. R Expression of GLUT1, HK2 and LDHA
Fig 4: Overexpression of MAP3K9 abated the suppressive effect of miR-1193 upregulation on the progression of CSCC cells. SCC13 and Colo16 cells were transfected with miR-NC, miR-1193, miR-1193 + vector, or miR-1193 + MAP3K9. (A) Western blot assay was utilized to analyze the protein expression of MAP3K9 (ANOVA). (B and C) Cell viability was assessed by MTT assay (ANOVA). (D) Colony formation ability was evaluated using colony formation assay (ANOVA). (E) Cell apoptosis was analyzed by flow cytometry analysis (ANOVA). (F and G) Transwell assay was employed to determine cell migration and invasion (100×) (ANOVA). (H and I) Lactate production or glucose consumption was measured by lactate assay kit or glucose assay kit, respectively (ANOVA). (J and K) ECAR was analyzed by Seahorse Bioscience XF24 extracellular flux analyzer (ANOVA). (L and M) Western blot assay was carried out to determine the protein levels of HK2 and LDHA (ANOVA). *P<0.05.
Fig 5: miR-216b inhibits BC cell proliferation, migration and invasion by targeting HK2. A, BC cell proliferation in response to miR-216b upregulation or HK2 overexpression assessed by CCK-8; B, BC cell migration detected by scratch test; C, BC cell invasion detected by Transwell assay; the experiment was repeated for three times; data were analyzed using one-way ANOVA. *, p < 0.05 compared with the cells transfected with mimic NC + oe-NC; #, p < 0.05 compared with miR-216b mimic +oe-NC.
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