Evidence for msiCAT-tailing on nuclear-encoded mitochondrial proteins in GBM cells

A. The expression levels of RQC genes changed significantly in GBM tumor tissues (N=153) and normal brain tissues (N=206). Significance was calculated by using unpaired student’s t-test, #, logFC (fold change) > 1, adj.P.Val < 0.001.

B. Western blot analysis of msiCAT-tailed mitochondrial proteins and RQC factors in invasive patient-derived GSC and control NSC cells. Actin serves as the loading control. Red arrow heads indicate short CAT-tailed mitochondrial proteins. “short” and “long” represent exposure time. Red numbers represent fold changes in protein levels compared to controls (here NSC), labeling of fold changes in the figures below follows the same rule.

C. Western blot analysis of 5×FLAG-tagged β-globin reporter proteins in GBM and control cells, showing that the RQC system of GBM cells generates more CAT-tailed proteins. Actin serves as the loading control. Red numbers represent fold changes in protein levels compared to the control (left lane: NHA, no anisomycin).

D. Western blot analysis of overexpressed ATP5⍺-AT3 and ATP5⍺-AT20 in GBM and control cells. GAPDH serves as the loading control. SVG, SVG p12 cells; NHA, normal human astrocytes; SF, SF268 cells; GSC, GSC827 cells; labeling of cell lines in the figures below follows the same rule. Ctrl, empty vector; ATP5⍺, non-AT-tailed protein; AT3, ATP5⍺-AT3; AT20, ATP5⍺-AT20; labeling of transgenes in the figures below follows the same rule. Arrowheads from bottom to top indicate endogenous ATP5⍺, ATP5⍺-AT3, ATP5⍺-AT20, oligomers and aggregates of msiCAT-tailed ATP5⍺ proteins.

E. Immunofluorescence staining shows that endogenous ATP5⍺ proteins can form aggregates in GBM cells. TOM20 (red) serves as the mitochondrial marker. Arrowheads in GBM cells indicate aggregates composed by ATP5⍺. In this and subsequent figures, the scale bar is indicated in the images.

F. Quantification of G. n=3, chi-squared test. ***, P < 0.001; ****, P < 0.0001. All data are representative of at least 3 independent experiments. The total number of cells counted is indicated in each column.

msiCAT-tailed ATP5⍺ proteins impair mitochondrial respiration but increase mitochondrial membrane potential in GBM cells

A. TMRM staining shows that patient-derived GSC (pGSC) cells display high mitochondrial membrane potential. n=3, unpaired student’s t-test. ***, P < 0.001; ****, P < 0.0001.

B. ATP measurement shows that patient-derived GSC (pGSC) cells has low mitochondrial ATP production. n=3, unpaired student’s t-test. **, P < 0.01; ***, P < 0.001.

C. JC-10 staining shows that genetic inhibition of msiCAT-tailing pathway reduces mitochondrial membrane potential in GBM but not in NHA control cells. n=3, unpaired student’s t-test. ****, P < 0.0001; ns, not significant.

D. JC-10 staining shows that ectopic expression of ATP5⍺-AT3 and ATP5⍺-AT20 increases mitochondrial membrane potential in GBM but not in control cells. n=3, unpaired student’s t-test. ****, P < 0.0001; ns, not significant.

E. BN-PAGE western blot for detecting ATP synthase assembly shows that ATP5⍺-AT3 is incorporated into the composition of complex V, but ATP5⍺-AT20 forms high molecular weight protein aggregates in mitochondria of GBM cells. SC: respiratory supercomplex; C-V: complex V/ATP synthase.

F, H. OCR indicates that the reduction of mitochondrial consumption rate of SF268 cells with ATP5⍺-AT3 and ATP5⍺-AT20 expression. Oligomycin (1.5 µM), FCCP (1.0 µM), and rotenone/antimycin A (R/A, 0.5 µM) were added sequentially to the reaction cells.

G, I. Statistical summary of mitochondrial respiration parameters in F and H, including non-mitochondrial respiration, basal respiration, maximum respiration, spare respiration, proton leaks and ATP production. n=3, unpaired student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

msiCAT-tailing product regulates mPTP status in GBM cells

A. Mitochondrial transition pore assay shows that GBM has reduced mPTP opening compared to NHA controls, which can be reversed by transient anisomycin treatment (200 nM).

B. Quantification of A. n=3, unpaired student’s t-test. ****, P < 0.0001.

C. Calcium retention capacity (CRC) of isolated mitochondria from cells measured by using Calcium Green-5N dye. NHA is used as the control cell line. Cyclosporin A (CsA, a mPTP inhibitor)-treated sample serves as a positive control.

D. Statistic of C. The data shows that GBM cells exhibited high CRC, whereas mitochondria from cells pretreated with anisomycin (200 nM) or with NEMF knockout (sgNEMF) are attenuated. n=3, unpaired student’s t-test. ***, P < 0.001; ****, P < 0.0001.

E. Mitochondrial transition pore assay shows that ectopic expression of ATP5⍺-AT3 and ATP5⍺-AT20 inhibits mPTP opening in GBM cells.

F. Quantification of E. n=3, unpaired student’s t-test. ****, P < 0.0001.

G. BN-PAGE western blot shows that the expression of ATP5⍺-AT3 and ATP5⍺-AT20 changes the pattern of ANT1/2 protein in GBM cells, missing a band (marked by a red * on the blot), and forming high molecular weight protein aggregates. SC: respiratory supercomplex; C-V: complex V/ATP synthase.

msiCAT-tailed ATP5⍺ protein promotes GBM progression

A. MTT assay shows that the expression of ATP5⍺-AT3 and ATP5⍺-AT20 promotes GBM cell proliferation. n=3, unpaired student’s t-test. **, P < 0.01; ***, P < 0.001.

B. MTT assay shows that the expression of ATP5⍺-AT3 and ATP5⍺-AT20 does not promote NHA cell proliferation. n=3, unpaired student’s t-test. ns, not significant.

C. Transwell assay shows that the expression of ATP5⍺-AT3 and ATP5⍺-AT20 promotes GBM cell (SF) but not control cell (NHA) migration.

D. Quantification of C showing the number of migrated cells. n=3, unpaired student’s t-test. ****, P < 0.0001; ns, not significant.

E. TUNEL staining shows that the expression of ATP5⍺-AT3 and ATP5⍺-AT20 can block STS (1 µM)-induced apoptosis in GBM cells. TUNEL-Cy3 was used to indicate apoptotic cells. DAPI was used to stain nucleus.

F. Quantification of E showing the percentage of TUNEL-positive cells in the population. n=3, unpaired student’s t-test. ****, P < 0.0001.

G. MTT assay shows that the expression of ATP5⍺-AT3 and ATP5⍺-AT20 enhances resistance to temozolomide (TMZ, 150 µM). TMZ treated SF-Ctrl group is used as the control. n=3, unpaired student’s t-test. ***, P < 0.001.

Inhibition of msiCAT-tailing impedes GBM progression

A. Cell viability assay shows that patient-derived GSC (pGSC) cells are significantly lower than control NSC cells, after 48 hours of anisomycin treatment. ***, P < 0.001; ****, P <0.0001, compared to the NSC controls at a certain dose of anisomycin.

B. Cell viability assay shows that genetic inhibition of msiCAT-tailing pathway hampers GBM cell proliferation. **, P < 0.01; ***, P < 0.001; ****, P <0.0001, compared to the control group at the corresponding time.

C. Transwell assay shows that genetic inhibition of msiCAT-tailing pathway blocks GBM cell (SF) but not control cell (NHA) migration.

D. Quantification of C showing the number of migrated cells. n=3, unpaired student’s t-test. ***, P < 0.001; ns, not significant.

E. JC-10 staining shows that pharmacological inhibition of msiCAT-tailing synthesis with anisomycin reduces mitochondrial membrane potential in GBM but not in NHA control cells. n=3, unpaired student’s t-test. ***, P < 0.001; ns, not significant.

F. Western blot analysis of PARP in anisomycin treated and untreated GSC cells shows that pharmacological inhibition of msiCAT-tailing pathway potentiates STS-induced apoptosis. Red numbers below the blots represent the ratio of cleaved PARP (c-PARP) to PARP protein. Actin serves as the loading control.

G. TUNEL staining shows that pharmacological inhibition of msiCAT-tailing pathway promotes STS (1 µM)-induced apoptosis in GBM cells. TUNEL-Cy3 is used to indicate apoptotic cells. DAPI is used to stain nucleus.

H. Quantification of G showing the percentage of TUNEL-positive cells in the population. n=3, unpaired student’s t-test. **** P < 0.0001. DMSO is used as the vehicle control.

I. Cell viability assay shows that pharmacological inhibition of msiCAT-tailing pathway could reduce the resistance of GBM cells to TMZ treatment (150 µM). n=3, unpaired student’s t-test. * P < 0.05; **, P < 0.01.

J. Neurosphere formation assay shows that pharmacological inhibition of msiCAT-tailing pathway can cooperate with TMZ to reduce the formation of spheroids in GBM cells, indicating the reduction of drug resistance.

K. Quantification of J. n=3, unpaired student’s t-test. **, P < 0.01.

A working model of how msiCAT-tail modified ATP5⍺ protein affects mitochondrial function in GBM cells

In normal cells, the ATP5⍺ protein encoded by the nuclear genome enters the mitochondrial matrix through a co-translational import mechanism through the TOM/TIM complex and is subsequently assembled into ATP synthase (Left Side). In GBM cells, the CAT-tailed ATP5⍺ protein forms insoluble aggregates close to the outer membrane of mitochondria or can be imported into mitochondria. In the mitochondrial matrix, proteins with shorter CAT-tails easily incorporate into ATP synthase and affect its function, which is manifested by a decrease in the ATP synthesis rate and accumulation of protons (H+), resulting in an increase in mitochondrial membrane potential. Ultimately, changes in ATP synthase leads to the malfunction of mPTP, thereby affecting cell proliferation, migration and resistance to drug-induced apoptosis (Right Side).

The RQC pathway is disturbed in GBM cells

A. Western blot analysis of some RQC factors in control (SVG, NHA) and GBM (SF268, GSC827) cell lines. Actin serves as the loading control. Red * indicates a modified form of NEMF in GSC827 cells. Red numbers represent fold changes in protein levels compared to controls (here SVG), labeling of fold changes in the figures below follows the same rule.

B. Immunofluorescence staining shows that Flag-tagged ATP5⍺-AT3 and ATP5⍺-AT20 (green) form aggregates in GBM and control cells. TOM20 (red) serves as the mitochondrial marker. In this and subsequent figures, the scale bar is indicated in the images.

C. Quantification of A, n=3, chi-squared test. ****, P < 0.0001. The total number of cells counted is indicated in each column.

Differential expression analysis for RQC genes in GBM patients and healthy controls

GBM cells have high mitochondrial membrane potentials but low mitochondrial ATP levels

A. JC-10 staining shows that GBM cells have high mitochondrial membrane potentials than NHA (control) cells. n=3, unpaired student’s t-test. ***, P < 0.001

B. BioTracker ATP-red dye staining shows that GBM cells have low mitochondrial ATP production than NHA (control) cells. MitoTracker Green is used to indicate mitochondrial mass.

C. Quantification of B. n=3, unpaired student’s t-test. ****, P < 0.0001.

D. Western blot analysis shows the efficiency of overexpression and knockdown in cells. Actin serves as the loading control.

The regulation of mPTP by CAT-tailing on ATP5⍺

A. Immunofluorescence staining shows that anisomycin (200 nM) treatment inhibits protein aggregation formed by endogenous ATP5⍺. TOM20 (red) serves as the mitochondrial marker.

B. Quantification of A, n=3, chi-squared test. *, P < 0.05. The total number of cells counted is indicated in each column.

C. Co-immunoprecipitation shows no direct interaction between ATP5⍺ and CypD or ANT1/2 can be found in GBM cells. Red arrowheads indicate target proteins.

D. Western blotting of cytosolic and isolated mitochondrial samples shows that expression of ATP5⍺-AT3 reduces CypD levels in GBM cells. TOM20 serves as the mitochondrial marker and loading control.

msiCAT-tailing regulates cell survival in GBM cells

A. Wound healing assay shows that the expression of ATP5⍺-AT3 and ATP5⍺-AT20 promotes GBM cell (SF268) migration.

B. Quantification of A showing the healing rate indicated by scratch wound coverage after 24 and 48 hours. n=3, unpaired student’s t-test. **, P < 0.01.

C. RT-PCR shows no significant increase of mitochondrial unfolded protein response markers. Actin serves as the loading control.

D. TUNEL staining shows that GBM cells are more resistant to STS (1 µM)-induced apoptosis than control cells. TUNEL-Cy3 is used to indicate apoptotic cells. DAPI is used to stain nucleus.

E. Quantification of C showing the percentage of TUNEL-positive cells in the population. n=3, unpaired student’s t-test. ***, P < 0.0001; ****, P < 0.0001.

F. Western blot analysis of PARP shows that GBM cells are more resistant to apoptosis induced by 30, 90, 180 min of STS (1 µM) treatment. Cleaved PARP serves as an indicator for apoptosis. Actin and GAPDH are used as loading controls. Red numbers below the blots represent the ratio of cleaved PARP (c-PARP) to PARP protein.

Inhibition of msiCAT-tailing promotes apoptosis

A. Cell viability assay shows that genetic inhibition of msiCAT-tailing pathway also hampers NHA cell proliferation. **, P < 0.01; ****, P <0.0001, compared to the control group at the corresponding time.

B. Transwell assay shows that pharmacological inhibition of msiCAT-tailing pathway blocks GSC cell (SF) but not control cell (NHA) migration.

C. Quantification of C showing the number of migrated cells. n=3, unpaired student’s t-test. ****, P < 0.0001; ns, not significant.

D. TUNEL staining shows that genetic inhibition of msiCAT-tailing pathway promotes STS (1 µM)-induced apoptosis in GBM cells. TUNEL-FITC is used to indicate apoptotic cells. DAPI is used to stain nucleus.

E. Quantification of G showing the percentage of TUNEL-positive cells in the population. n=3, unpaired student’s t-test. **** P < 0.0001. DMSO is used as the vehicle control.