Fig 2: PSMD14 inhibitor Thiolutin improves chemotherapy in HNSCC. (A) The chemical structure of Thiolutin. (B) The IC50 of Thiolutin in SCC15 and UM1 cell lines was measured. (C) The abundance of E2F1/Akt/SOX2 axis modulated by PSMD14 and proapoptotic markers was probed in SCC15 and UM1 cells treated with Thiolutin for 24 hours. (D) The cell viability of HNSCC cells was extremely inhibited by Thiolutin treatment. (E) THL attenuated the colony formation of SCC15 and UM1 cells. (F-H) The measurement of cisplatin IC50 (F) and flow cytometry (G and H) showed that THL significantly increased sensitivity of HNSCC cells to cisplatin. (I) The apoptosis rates of HNSCC cells treated with THL, CDDP and THL plus CDDP were measured by using flow cytometry. The concentration of THL in (C-I) was 0.6 µM in SCC15 cells and 0.3 µM in UM1 cells, respectively. (J) The mice (n = 5) with xenografts were administered with saline, THL (0.75 mg/kg), CDDP (2.5 mg/kg) or CDDP (2.5 mg/kg) combined with THL (0.75 mg/kg) every 3 days until sacrifice. Then the representative photos of indicated treated HNSCC xenografts were presented. The size and weight of tumors were significantly reduced in combined treatment group. (K) The expression of Ki67 and cleaved Caspase-3 was measured by IHC staining among four groups described in (J). Scale bar, 100 µm. Data in this figure, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. THL, Thiolutin. CDDP, cisplatin

Fig 3: Effects of a2M on the expression of pluripotency-associated genes in irradiated hBMMSCs. Reverse transcription-quantitative polymerase chain reaction was employed to detect the effects of a2M on the mRNA expression levels of (A) Sox2 and (B) Nanog in hBMMSCs at 24 h following radiation. (C and D) Western blotting was employed to detect the effects of a2M on the protein expression levels of Sox2 and Nanog in hBMMSCs at 24 h following radiation. *P<0.05 vs. blank control group, #P<0.05 vs. radiation control group (n=3). hBMMSC, human bone marrow mesenchymal stem cells; a2M, a-2-macroglobulin; Sox2, sex determining region Y.

Fig 4: Proliferation of PDLSCs cultured on B-dECM, P-dECM and no-ECM. (A) Representative images of passage 2 PDLSCs cultured on B-dECM, P-dECM and no-ECM after day 1, day 4, and day 7 to monitor cell density and proliferative ability. White box, high magnification of PDLSCs in different cultured ECMs. Black bar, 200 µm. (B) Number of PDLSCs on specific dECMs and no-ECM at day 1, 4 and 7. (C, D) Changes in cell cycle after expansion for 24 h were measured using flow cytometry. Analytical comparison of cell cycle differences between groups. (E) mRNA expression levels of Sox-2, Nanog, and Oct-4 to assess stemness and Ki67 and Cyclin D1 to assess proliferative ability of PDLSCs cultured on specific dECMs and no-ECM. (F) Western blotting results showing the expression of stemness- and proliferation-related proteins in PDLSCs cultured on specific dECMs and no-ECM. Quantitation of blots (right panel). (G) Intracellular ROS accumulation in PDLSCs cultured on dECMs and no-ECM, labeled with DCF-DA; fluorescence intensity was measured by flow cytometry. The data are presented as the means ± SD; n = 5. * P < 0.05, ** P < 0.01 and *** P < 0.001 represent significant differences in the indicated columns (P-dECM and B-dECM) compared with the no-ECM group. ## P < 0.01 and ### P < 0.001 represent significant differences between the P-dECM and B-dECM groups.

Fig 5: p97 inhibition reduces breast cancer growth, the CSC population, and mammosphere formation.a MDA-MB-231 cells were treated with 1.25, 2.5, 5, and 10 µM Eer I and NMS-873 for 24 h. DMSO was used as a vehicle control. The ubiquitinated proteins in cell lysates were analyzed by immunoblotting. GAPDH was used as a loading control. b Proliferation of MCF-7 and MDA-MB-231 cells treated with Eer I and NMS-873, with IC50 values indicated. c MCF10A, MCF-7, and MDA-MB-231 cells were treated with 1.25, 2.5, 5, and 10 µM Eer I for 24 h and cell viability was measured. d Growth of orthotopic tumors formed by MDA-MB-231 cells and treated with p97 inhibitors or DMSO. e Growth of orthotopic tumors formed by ALDH+ CSCs and ALDH- non-CSC cells and treated with p97 inhibitors or DMSO. f, g qPCR and immunoblotting analysis of p97, OCT4, and SOX2 in adherent and spheroid MDA-MB-231 cells. ß-actin serves as a loading control for immunoblotting. h Twenty-four hours after MDA-MB-231 cells were seeded, Eer I and NMS-873 were added to the culture at 0.1, 0.4, 1.6, and 6.4 µM for 7 days to assess their impact on mammosphere formation. DMSO was used as a vehicle control. Left: representative images of the spheres treated with 6.4 µM Eer I or 6.4 µM NMS-873. Bar: 100 µm. Right: the percentage of spheres formed under each condition relative to the control group, with IC50 values indicated. i Eer I and NMS-873 were added to the culture at 0.1, 0.25, 0.5, and 1 µM for 7 days after the secondary spheres were formed to assess their impact on mammosphere maintenance. Left: representative images of the spheres treated with 1 µM Eer I or 1 µM NMS-873. Bar: 100 µm. Right: the percentage of spheres formed under each condition relative to the control group, with IC50 values indicated. Data were shown as mean + or ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.