human origin cal33  (ATCC)

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 promotes mitochondrial fragmentation. (A) Representative confocal microscopy images of empty vector- and PPA2-expressing stable CAL33, FaDu, BJ and HaCaT cells showing mitochondrial morphology, and (B) the ImageJ-based analysis of mitochondrial branch length, calculated from images with multiple cells randomly taken for each sample of three different experiments. (C) Representative confocal images showing the mitochondrial morphology in the shControl, sh PPA2 , and sh PPA2 cells with transient overexpression of PPA2 or PPA2ΔMLS and (D), The ImageJ-based analysis of mitochondrial branch length. (E) Representative confocal images showing the mitochondrial morphology in the shControl, sh PPA2 and sh PPA2 cells with transient overexpression of PPA2 or PPA2ΔMLS and (F), the ImageJ-based analysis of mitochondrial branch length. Scale bars: 20 μm. The number of cells analyzed per condition is > 25. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Confocal Microscopy, Plasmid Preparation, Expressing, Over Expression

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 promotes DNM1L phosphorylation at S616 and mitochondrial translocation to induce mitochondrial fission. (A) Western blots show the expression of p-DNM1L S616, DNM1L, and V5 along with ACTB as a loading control in empty vector- and PPA2-V5 expressing stable CAL33, FaDu, and BJ cells. (B) Western blots showing the expression of p-DNM1L, GAPDH, and TOMM20 in the mitochondrial and cytosolic fractions in empty vector- and PPA2-V5 expressing stable FaDu cells. (C) Western blots confirming the knockdown of DNM1L. (D) Representative confocal microscopy images showing mitochondrial morphology in control and DNM1L-knockdown cells, and (E), the ImageJ-based analysis of mitochondrial branch length. (F) Western blots showing the expression of p-DNM1L S616, DNM1L, and V5 along with ACTB as a loading control in shControl, sh PPA2 and sh PPA2 cells with transient overexpression of PPA2 or PPA2ΔMLS. (G) Representative confocal images of FaDu cells showing the colocalization of TOMM20 and p-DNM1L, and (H), the ImageJ-based quantification of p-DNM1L S616 colocalization with TOMM20. (I) western blots showing the expression of p-DNM1L S616, DNM1L, and V5 along with ACTB as a loading control in shControl, sh PPA2 and sh PPA2 cells with transient overexpression of PPA2 or PPA2R127A. (J) Representative confocal images of FaDu cells showing the colocalization of TOMM20 and p-DNM1L, and (K) the ImageJ-based quantification of p-DNM1L S616 colocalization with TOMM20. Scale bars: 20 μm. For D, G, J panels, the number of cells analyzed per condition is > 25. Error bars: mean ±S.D., n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Expressing, Control, Plasmid Preparation, Knockdown, Confocal Microscopy, Over Expression

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 induces symmetric mitochondrial fission through the MFF-DNM1L axis. (A) western blots show the expression of MFF, FIS1, and PPA2-V5 along with ACTB as a loading control in empty vector- and PPA2-expressing stable CAL33, FaDu and BJ cells. (B) Representative confocal images of control and PPA2-overexpressing FaDu cells showing the colocalization of TOMM20 and MFF. (C) quantification of the MFF fluorescence intensity. (D) quantitative real-time PCR-based analysis of relative mtDNA content in vector- and PPA2-expressing stable FaDu cells. (E) Representative confocal images showing the colocalization of mitochondrial marker COX4 with mtDNA. (F) Quantitative real-time PCR-based analysis of relative mtDNA content in control or PPA2-overexpressing FaDu cells transfected with mock or TFAM gDNA. (G) Western blots showing the expression of p-DNM1L S616, DNM1L, MFF, MTFP1, COX4, TFAM, and V5 along with ACTB as loading in control or PPA2-overexpressing FaDu cells transfected with mock or TFAM gDNA. (H) Representative confocal images showing the colocalization of p-DNM1L S616 and TOMM20 along with mitochondrial morphology in control or PPA2-overexpressing FaDu cells transfected with mock or TFAM gDNA. (I) ImageJ-based analysis of mitochondrial branch length; the number of cells analyzed per condition is > 25. (J) Quantitative real-time PCR-based analysis of relative mtDNA content in control or PPA2-overexpressing FaDu cells transfected with shControl or sh MTFP1 . (K) Western blots showing the expression of MFF, V5, and MTFP1 along with ACTB as a loading control in control or PPA2-overexpressing FaDu cells transfected with shControl or sh MTFP1 . (L and M) Western blots showing the expression of MFF, V5, and MTFP1 along with ACTB as a loading control in shControl, sh PPA2 , and sh PPA2 cells with (L) transient overexpression of PPA2 or PPA2ΔMLS or (M) transient overexpression of PPA2 or PPA2 R127A . The number of cells analyzed per condition in the C and I panel is > 25. Scale bars: 20 μm. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Fluorescence, Real-time Polymerase Chain Reaction, Marker, Transfection, Over Expression

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2-induced symmetric mitochondrial fission leads to mitochondrial proliferation. (A) western blots show the expression of COX4, PPA2-V5 along with ACTB as a loading control in empty vector- and PPA2-expressing stable CAL33, FaDu and BJ cells, and (B), the mean fluorescence intensity (MFI) of MitoTracker green obtained by flow cytometry and normalized to the control in control and PPA2-overexpressing CAL33, FaDu and BJ cells. (C) the cellular ATP level representing relative light units (RLU) in control and PPA2-overexpressing CAL33, FaDu and BJ cells. (D) western blots showing the expression of PPARGC1A, COX4, and V5 along with ACTB as a loading control in empty vector- and PPA2-expressing stable FaDu cells. (E-F) Representative confocal images of rtTA3-expressing FaDu cells transfected with MitoTimer indicating the old mitochondria (red) and new mitochondria (green) (E), and the ImageJ-based quantification of green:red ratio indicating the occurrence of mitochondrial biogenesis (F). The (G-J) western blots showing the expression of COX4, and V5 along with ACTB as a loading control in shControl, sh PPA2 and sh PPA2 cells with (G) transient overexpression of PPA2 or PPA2ΔMLS, (H) quantification of COX4:ACTB expression normalized to control or (I) transient overexpression of PPA2 or PPA2 R127A (J) quantification of COX4:ACTB expression normalized to control. (K and L) western blots showing the expression of COX4 and V5 along with ACTB as a loading control in control or PPA2-overexpressing FaDu cells transfected with shControl, (K) sh DNM1L or (L) sh MTFP1 . (M, N) western blots showing the expression of COX4 along with ACTB as a loading control in the MTFP1 rescue set (M), quantification of COX4:ACTB expression normalized to control (N). Scale bars: 20 μm. Error bars: mean ±S.D., n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Fluorescence, Flow Cytometry, Transfection, Over Expression

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 induces asymmetric mitochondrial fission during mitochondrial stress. (A) western blots showing the expression of p-DNM1L S616, DNM1L, FIS1, MFF, MTFP1, COX4, and PPA2-V5 along with ACTB in the control or PPA2-overexpressing CAL33 and FaDu cells treated with CCCP (10 µM, four h in all panels). (B) Representative confocal images showing the mitochondrial morphology of the control or PPA2-overexpressing FaDu cells treated with CCCP, and (C) ImageJ-based analysis of mitochondrial branch length; the number of cells analyzed per condition is > 25. (D) Representative confocal images showing the colocalization of p-DNM1L and TOMM20 in control or PPA2-overexpressing FaDu cells treated with CCCP, and (E), ImageJ-based quantification of p-DNM1L S616 colocalization with TOMM20. (F) Representative confocal images of control or PPA2-overexpressing FaDu cells treated with CCCP, showing the colocalization of MFF-TOMM20, and (G), ImageJ-based quantification of MFF colocalization with TOMM20. (H) Representative confocal images of control or PPA2-overexpressing FaDu cells treated with CCCP, showing the colocalization of FIS1-TOMM20, and (I), ImageJ-based quantification of FIS1 colocalization with TOMM20. (J) Representative confocal images of control or PPA2-overexpressing FaDu cells treated with CCCP, showing the apposition of LAMP1-TOMM20, and (K) ImageJ-based quantification of the number of mitochondrial clusters apposed to LAMP1. (L) quantitative real-time PCR-based analysis of relative mtDNA content of control or PPA2-overexpressing FaDu cells in the presence of CCCP. Scale bars: 20 μm. The number of cells analyzed per condition in the C, E, G, I, and K panels is > 25. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: Mitochondrial fission is compromised in PPA2-knockdown cells. (A and B) western blots showing the expression of p-DNM1L S616, DNM1L, FIS1, MTFP1, and PPA2-V5 along with ACTB in the shControl or sh PPA2 -expressing (A) CAL33 and (B) FaDu cells treated with CCCP (10 µM, 4 h in all panels). (C and E) Representative confocal images showing the mitochondrial morphology in the shControl or sh PPA2 -expressing (C) CAL33 and (D) FaDu cells treated with CCCP, and (D and F) ImageJ-based analysis of mitochondrial branch length; number of cells analyzed per condition is > 25. (G and I) Representative confocal images showing the colocalization of p-DNM1L and TOMM20 in the shControl or sh PPA2 -expressing (G) CAL33 and (I) FaDu cells treated with CCCP, and (H and J) quantification of p-DNM1L S616 colocalization with TOMM20 normalized to the control; the number of cells analyzed per condition is > 25. (K) quantitative real-time PCR-based analysis of relative mtDNA content in FaDu cells expressing shControl or sh PPA2 . Scale bars: 20 μm. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Knockdown, Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 regulates the elimination of fragmented mitochondria through mitophagy during mitochondrial stress. (A and B) western blots showing the expression of COX4 along with ACTB in control or PPA2-overexpressing (A) CAL33 and (B) FaDu cells treated with CCCP (10 µM, four h in all panels). (C) Representative confocal images showing the colocalization of TOMM20 and LC3 in the control or PPA2-overexpressing FaDu cells treated with CCCP and (D) quantification of the mitochondria colocalized with LC3 (Pearson’s co-efficient). (E) confocal live cell imaging of control and PPA2-overexpressing FaDu cells transfected with mito-mKeima followed by CCCP treatment, and (F) ImageJ-based quantification of red:green ratio. (G and H) western blots showing the expression of COX4 along with ACTB in the shControl and sh PPA2 -expressing stable (G) CAL33 and (H) and FaDu cells treated with CCCP. (I) ImageJ-based quantification of the mitochondria colocalized with LC3 (Pearson’s co-efficient) in shControl or sh PPA2 -expressing FaDu cells treated with CCCP. (J) ImageJ-based quantification of red:green ratio in control and PPA2-overexpressing FaDu cells transfected with mito-mKeima followed by CCCP treatment. (K) ImageJ-based quantification of the mitochondria colocalized with LC3 (Pearson’s co-efficient) in the FaDu cells expressing vector, PPA2 or PPA2 R127A following CCCP treatment. (L) western blots showing the expression of COX4 and V5 along with ACTB in the shControl and sh PPA2 -expressing stable FaDu cells transiently expressing PPA2 or PPA2 R127A in the presence of CCCP. (M) western blots showing the expression of COX4, ATG5, and V5 along with ACTB in shControl or sh ATG5 -expressing stable FaDu cells co-expressing vector or PPA2 in the presence of CCCP. (N) western blots showing the expression of COX4, BECN1, and V5 along with ACTB in shControl or sh BECN1 -expressing stable FaDu cells co-expressing vector or PPA2 in the presence of CCCP. Scale bars: 20 μm. For D, F, I, J, K the number of cells analyzed per condition is > 25. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Western Blot, Expressing, Control, Live Cell Imaging, Transfection, Plasmid Preparation

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 promotes mitochondrial fragmentation. (A) Representative confocal microscopy images of empty vector- and PPA2-expressing stable CAL33, FaDu, BJ and HaCaT cells showing mitochondrial morphology, and (B) the ImageJ-based analysis of mitochondrial branch length, calculated from images with multiple cells randomly taken for each sample of three different experiments. (C) Representative confocal images showing the mitochondrial morphology in the shControl, sh PPA2 , and sh PPA2 cells with transient overexpression of PPA2 or PPA2ΔMLS and (D), The ImageJ-based analysis of mitochondrial branch length. (E) Representative confocal images showing the mitochondrial morphology in the shControl, sh PPA2 and sh PPA2 cells with transient overexpression of PPA2 or PPA2ΔMLS and (F), the ImageJ-based analysis of mitochondrial branch length. Scale bars: 20 μm. The number of cells analyzed per condition is > 25. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Confocal Microscopy, Plasmid Preparation, Expressing, Over Expression

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 promotes DNM1L phosphorylation at S616 and mitochondrial translocation to induce mitochondrial fission. (A) Western blots show the expression of p-DNM1L S616, DNM1L, and V5 along with ACTB as a loading control in empty vector- and PPA2-V5 expressing stable CAL33, FaDu, and BJ cells. (B) Western blots showing the expression of p-DNM1L, GAPDH, and TOMM20 in the mitochondrial and cytosolic fractions in empty vector- and PPA2-V5 expressing stable FaDu cells. (C) Western blots confirming the knockdown of DNM1L. (D) Representative confocal microscopy images showing mitochondrial morphology in control and DNM1L-knockdown cells, and (E), the ImageJ-based analysis of mitochondrial branch length. (F) Western blots showing the expression of p-DNM1L S616, DNM1L, and V5 along with ACTB as a loading control in shControl, sh PPA2 and sh PPA2 cells with transient overexpression of PPA2 or PPA2ΔMLS. (G) Representative confocal images of FaDu cells showing the colocalization of TOMM20 and p-DNM1L, and (H), the ImageJ-based quantification of p-DNM1L S616 colocalization with TOMM20. (I) western blots showing the expression of p-DNM1L S616, DNM1L, and V5 along with ACTB as a loading control in shControl, sh PPA2 and sh PPA2 cells with transient overexpression of PPA2 or PPA2R127A. (J) Representative confocal images of FaDu cells showing the colocalization of TOMM20 and p-DNM1L, and (K) the ImageJ-based quantification of p-DNM1L S616 colocalization with TOMM20. Scale bars: 20 μm. For D, G, J panels, the number of cells analyzed per condition is > 25. Error bars: mean ±S.D., n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Expressing, Control, Plasmid Preparation, Knockdown, Confocal Microscopy, Over Expression

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 induces symmetric mitochondrial fission through the MFF-DNM1L axis. (A) western blots show the expression of MFF, FIS1, and PPA2-V5 along with ACTB as a loading control in empty vector- and PPA2-expressing stable CAL33, FaDu and BJ cells. (B) Representative confocal images of control and PPA2-overexpressing FaDu cells showing the colocalization of TOMM20 and MFF. (C) quantification of the MFF fluorescence intensity. (D) quantitative real-time PCR-based analysis of relative mtDNA content in vector- and PPA2-expressing stable FaDu cells. (E) Representative confocal images showing the colocalization of mitochondrial marker COX4 with mtDNA. (F) Quantitative real-time PCR-based analysis of relative mtDNA content in control or PPA2-overexpressing FaDu cells transfected with mock or TFAM gDNA. (G) Western blots showing the expression of p-DNM1L S616, DNM1L, MFF, MTFP1, COX4, TFAM, and V5 along with ACTB as loading in control or PPA2-overexpressing FaDu cells transfected with mock or TFAM gDNA. (H) Representative confocal images showing the colocalization of p-DNM1L S616 and TOMM20 along with mitochondrial morphology in control or PPA2-overexpressing FaDu cells transfected with mock or TFAM gDNA. (I) ImageJ-based analysis of mitochondrial branch length; the number of cells analyzed per condition is > 25. (J) Quantitative real-time PCR-based analysis of relative mtDNA content in control or PPA2-overexpressing FaDu cells transfected with shControl or sh MTFP1 . (K) Western blots showing the expression of MFF, V5, and MTFP1 along with ACTB as a loading control in control or PPA2-overexpressing FaDu cells transfected with shControl or sh MTFP1 . (L and M) Western blots showing the expression of MFF, V5, and MTFP1 along with ACTB as a loading control in shControl, sh PPA2 , and sh PPA2 cells with (L) transient overexpression of PPA2 or PPA2ΔMLS or (M) transient overexpression of PPA2 or PPA2 R127A . The number of cells analyzed per condition in the C and I panel is > 25. Scale bars: 20 μm. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Fluorescence, Real-time Polymerase Chain Reaction, Marker, Transfection, Over Expression

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2-induced symmetric mitochondrial fission leads to mitochondrial proliferation. (A) western blots show the expression of COX4, PPA2-V5 along with ACTB as a loading control in empty vector- and PPA2-expressing stable CAL33, FaDu and BJ cells, and (B), the mean fluorescence intensity (MFI) of MitoTracker green obtained by flow cytometry and normalized to the control in control and PPA2-overexpressing CAL33, FaDu and BJ cells. (C) the cellular ATP level representing relative light units (RLU) in control and PPA2-overexpressing CAL33, FaDu and BJ cells. (D) western blots showing the expression of PPARGC1A, COX4, and V5 along with ACTB as a loading control in empty vector- and PPA2-expressing stable FaDu cells. (E-F) Representative confocal images of rtTA3-expressing FaDu cells transfected with MitoTimer indicating the old mitochondria (red) and new mitochondria (green) (E), and the ImageJ-based quantification of green:red ratio indicating the occurrence of mitochondrial biogenesis (F). The (G-J) western blots showing the expression of COX4, and V5 along with ACTB as a loading control in shControl, sh PPA2 and sh PPA2 cells with (G) transient overexpression of PPA2 or PPA2ΔMLS, (H) quantification of COX4:ACTB expression normalized to control or (I) transient overexpression of PPA2 or PPA2 R127A (J) quantification of COX4:ACTB expression normalized to control. (K and L) western blots showing the expression of COX4 and V5 along with ACTB as a loading control in control or PPA2-overexpressing FaDu cells transfected with shControl, (K) sh DNM1L or (L) sh MTFP1 . (M, N) western blots showing the expression of COX4 along with ACTB as a loading control in the MTFP1 rescue set (M), quantification of COX4:ACTB expression normalized to control (N). Scale bars: 20 μm. Error bars: mean ±S.D., n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Fluorescence, Flow Cytometry, Transfection, Over Expression

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 induces asymmetric mitochondrial fission during mitochondrial stress. (A) western blots showing the expression of p-DNM1L S616, DNM1L, FIS1, MFF, MTFP1, COX4, and PPA2-V5 along with ACTB in the control or PPA2-overexpressing CAL33 and FaDu cells treated with CCCP (10 µM, four h in all panels). (B) Representative confocal images showing the mitochondrial morphology of the control or PPA2-overexpressing FaDu cells treated with CCCP, and (C) ImageJ-based analysis of mitochondrial branch length; the number of cells analyzed per condition is > 25. (D) Representative confocal images showing the colocalization of p-DNM1L and TOMM20 in control or PPA2-overexpressing FaDu cells treated with CCCP, and (E), ImageJ-based quantification of p-DNM1L S616 colocalization with TOMM20. (F) Representative confocal images of control or PPA2-overexpressing FaDu cells treated with CCCP, showing the colocalization of MFF-TOMM20, and (G), ImageJ-based quantification of MFF colocalization with TOMM20. (H) Representative confocal images of control or PPA2-overexpressing FaDu cells treated with CCCP, showing the colocalization of FIS1-TOMM20, and (I), ImageJ-based quantification of FIS1 colocalization with TOMM20. (J) Representative confocal images of control or PPA2-overexpressing FaDu cells treated with CCCP, showing the apposition of LAMP1-TOMM20, and (K) ImageJ-based quantification of the number of mitochondrial clusters apposed to LAMP1. (L) quantitative real-time PCR-based analysis of relative mtDNA content of control or PPA2-overexpressing FaDu cells in the presence of CCCP. Scale bars: 20 μm. The number of cells analyzed per condition in the C, E, G, I, and K panels is > 25. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: Mitochondrial fission is compromised in PPA2-knockdown cells. (A and B) western blots showing the expression of p-DNM1L S616, DNM1L, FIS1, MTFP1, and PPA2-V5 along with ACTB in the shControl or sh PPA2 -expressing (A) CAL33 and (B) FaDu cells treated with CCCP (10 µM, 4 h in all panels). (C and E) Representative confocal images showing the mitochondrial morphology in the shControl or sh PPA2 -expressing (C) CAL33 and (D) FaDu cells treated with CCCP, and (D and F) ImageJ-based analysis of mitochondrial branch length; number of cells analyzed per condition is > 25. (G and I) Representative confocal images showing the colocalization of p-DNM1L and TOMM20 in the shControl or sh PPA2 -expressing (G) CAL33 and (I) FaDu cells treated with CCCP, and (H and J) quantification of p-DNM1L S616 colocalization with TOMM20 normalized to the control; the number of cells analyzed per condition is > 25. (K) quantitative real-time PCR-based analysis of relative mtDNA content in FaDu cells expressing shControl or sh PPA2 . Scale bars: 20 μm. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Knockdown, Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Autophagy

Article Title: PPA2 activates MTFP1-DNM1L fission signaling to govern mitochondrial proliferation and mitophagy

doi: 10.1080/15548627.2025.2552900

Figure Lengend Snippet: PPA2 regulates the elimination of fragmented mitochondria through mitophagy during mitochondrial stress. (A and B) western blots showing the expression of COX4 along with ACTB in control or PPA2-overexpressing (A) CAL33 and (B) FaDu cells treated with CCCP (10 µM, four h in all panels). (C) Representative confocal images showing the colocalization of TOMM20 and LC3 in the control or PPA2-overexpressing FaDu cells treated with CCCP and (D) quantification of the mitochondria colocalized with LC3 (Pearson’s co-efficient). (E) confocal live cell imaging of control and PPA2-overexpressing FaDu cells transfected with mito-mKeima followed by CCCP treatment, and (F) ImageJ-based quantification of red:green ratio. (G and H) western blots showing the expression of COX4 along with ACTB in the shControl and sh PPA2 -expressing stable (G) CAL33 and (H) and FaDu cells treated with CCCP. (I) ImageJ-based quantification of the mitochondria colocalized with LC3 (Pearson’s co-efficient) in shControl or sh PPA2 -expressing FaDu cells treated with CCCP. (J) ImageJ-based quantification of red:green ratio in control and PPA2-overexpressing FaDu cells transfected with mito-mKeima followed by CCCP treatment. (K) ImageJ-based quantification of the mitochondria colocalized with LC3 (Pearson’s co-efficient) in the FaDu cells expressing vector, PPA2 or PPA2 R127A following CCCP treatment. (L) western blots showing the expression of COX4 and V5 along with ACTB in the shControl and sh PPA2 -expressing stable FaDu cells transiently expressing PPA2 or PPA2 R127A in the presence of CCCP. (M) western blots showing the expression of COX4, ATG5, and V5 along with ACTB in shControl or sh ATG5 -expressing stable FaDu cells co-expressing vector or PPA2 in the presence of CCCP. (N) western blots showing the expression of COX4, BECN1, and V5 along with ACTB in shControl or sh BECN1 -expressing stable FaDu cells co-expressing vector or PPA2 in the presence of CCCP. Scale bars: 20 μm. For D, F, I, J, K the number of cells analyzed per condition is > 25. Error bars: mean ±S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The oral cancer cell lines of human origin CAL33 (DSMZ, ACC-447) and FaDu (ATCC, HTB-43) were generously provided by Dr Goutam Sethi (National University of Singapore) and cultured in DMEM (Himedia, AL151A) and MEM (Himedia, AL047), respectively, with 10% FBS (Gibco 10270106) and antibiotic-antimycotic (1X; Himedia, A002A).

Techniques: Western Blot, Expressing, Control, Live Cell Imaging, Transfection, Plasmid Preparation