Journal: Nature Communications

Article Title: Transiently formed nucleus-to-cilium microtubule arrays mediate senescence initiation in a KIFC3-dependent manner

doi: 10.1038/s41467-024-52363-w

Figure Lengend Snippet: a Western blot detecting KIFC3 and senescence markers in RCTE cells with or without IR exposure. Three experiments were repeated independently with similar results. Immunofluorescent images showing KIFC3 (green) and polyglutamylated sinc-MTs (labeled with GT335 antibody, red) ( b ) and relative intensity of KIFC3 ( c ) in RCTE cells with or without IR exposure. CENTRIN2 (cyan) labels the ciliary base . n = 50 cells. Scale bar, 10 μm. SIM series section ( d ) and 3D surface-rendering reconstruction ( e ). Scale bar, 10 μm. Localization of KIFC3 (green) along polyglutamylated sinc-MTs (red) in RCTE cells exposed to IR. f Immunofluorescence images showing KIFC3 and sinc-MTs (labeled with α-tubulin) in control or siTTLL5 RCTE cells after IR exposure. CENTRIN2 labels the ciliary base. Scale bar, 10 μm. Western blot of senescence markers ( g ), quantitation of relative protein levels of senescence markers ( h ), SA-β-gal staining ( i ), relative mRNA level of SASP genes ( j ) in control or shKIFC3 RCTE cells at day 10 after IR exposure. Scale bar, 50 μm. All results from n = 3 independent experiments. Data are the mean ± SEM. Statistical significance was determined using one-way ANOVA. Three experiments were repeated independently with similar results ( d – f ). Source data are provided as a file.

Article Snippet: Sub-cloning templates of human CENEXIN1 and KIFC3 were purchased from Addgene (#73334) and DNASU(#HsCD00442644), and ODF2(iso6) was kindly provided by Dr. Kyung Lee (NIH/NCI).

Techniques: Western Blot, Labeling, Immunofluorescence, Control, Quantitation Assay, Staining

Journal: Nature Communications

Article Title: Transiently formed nucleus-to-cilium microtubule arrays mediate senescence initiation in a KIFC3-dependent manner

doi: 10.1038/s41467-024-52363-w

Figure Lengend Snippet: a Immunoprecipitation showing no interaction between V5-tagged KIFC3 and Myc-tagged FBF1 when overexpressed in 293 T cells. b , c Immunoprecipitation of endogenous CENEXIN1 with Myc-tagged FBF1 in IR-treated RCTE cells. d V5-tagged KIFC3 immunoprecipitates with EGFP-tagged CENEXIN1 when overexpressed in 293 T cells. e , f Endogenous CENEXIN1 immunoprecipitates with V5-tagged KIFC3 in control or IR-treated RCTE cells. g Immunofluorescence images showing PML-NBs translocation of CENEXIN1 (green) in IR-treated RCTE cells. PML (red) labels the PML-NBs. Scale bar, 10 μm. h 3D surface-rendering reconstruction of SIM section images showing the spatial relationship among CENEXIN1 (green), KIFC3 (red), and EB1 (cyan) labeled plus-ends of sinc-MTs at ciliary base in IR-treated RCTE cells. Scale bar, 10 μm. Three experiments were repeated independently with similar results ( a – h ). Source data are provided as a file.

Article Snippet: Sub-cloning templates of human CENEXIN1 and KIFC3 were purchased from Addgene (#73334) and DNASU(#HsCD00442644), and ODF2(iso6) was kindly provided by Dr. Kyung Lee (NIH/NCI).

Techniques: Immunoprecipitation, Control, Immunofluorescence, Translocation Assay, Labeling

Journal: Nature Communications

Article Title: Transiently formed nucleus-to-cilium microtubule arrays mediate senescence initiation in a KIFC3-dependent manner

doi: 10.1038/s41467-024-52363-w

Figure Lengend Snippet: a Diagram of WT and KIFC3 variants used in experiments. b Endogenous CENEXIN1 immunoprecipitates with V5-tagged WT or KIFC3 DN in RCTE cells, asterisk labels KIFC3 ∆N . c Immunofluorescent images showing localization of NeonGreen(NG)-tagged KIFC3 truncation variants (green) in RCTE cells. γ-tubulin (red) labels the ciliary base. Scale bar, 10 μm. Immunofluorescence images showing the effect of re-expression of KIFC3 or KIFC3 DN on CENEXIN1 and FBF1 translocation. CENEXIN1 (green), FBF1 (red) and PML (cyan) were immunostained by antibodies, respectively ( d ) and quantitation of PML-NBs ( e ) in shKIFC3 RCTE cells with or without IR exposure ( n = 40 cells). Scale bar, 10 μm. Immunofluorescence images showing the changes of CENEXIN1 (red) and PML (cyan) in control or over-expression NG-tagged KIFC3 DN RCTE cells with or without IR treatment ( f ) and quantitation of PML-NBs numbers per cell ( n = 40 cells) ( g ). Localization of KIFC3 DN was shown by NG direct fluorescence (green). Scale bar, 10 μm. h SA-β-gal staining of IR-treated shKIFC3 RCTE cells re-expressing KIFC3 or KIFC3 DN . Scale bar, 100 μm. i Immunofluorescence images showing the impact on the PML-NBs translocation of CENEXIN1 (green) and FBF1 (red) after re-expressing CEP170C-tagged KIFC3 in IR-treated shKIFC3 RCTE cells. γ-tubulin (cyan) labels the ciliary base. YFP direct fluorescence shown CEP170C-tagged KIFC3. Scale bar, 10 μm. All results from n = 3 independent experiments. Data are the mean ± SEM. Statistical significance was determined using one-way ANOVA. Three experiments were repeated independently with similar results ( b , c , h , i ). Source data are provided as a file.

Article Snippet: Sub-cloning templates of human CENEXIN1 and KIFC3 were purchased from Addgene (#73334) and DNASU(#HsCD00442644), and ODF2(iso6) was kindly provided by Dr. Kyung Lee (NIH/NCI).

Techniques: Immunofluorescence, Expressing, Translocation Assay, Quantitation Assay, Control, Over Expression, Fluorescence, Staining

Journal: Nature Communications

Article Title: Transiently formed nucleus-to-cilium microtubule arrays mediate senescence initiation in a KIFC3-dependent manner

doi: 10.1038/s41467-024-52363-w

Figure Lengend Snippet: a – d SA-β-gal staining ( a ), quantitation of SA-β-gal-positive cells ( n = 3 independent experiments, 6-8fields per experiment, 200 cells per field) ( b ) in control or siCENEXIN1 RCTE cells with or without IR exposure. Western blot of senescence markers ( c ), and relative mRNA level of SASP genes ( d ) in control or shCENEXIN1 RCTE cells with or without IR exposure. For IR treatment, cells were collected at day 10 after irradiation. Scale bar, 100 μm. SA-β-gal staining ( e ) and quantitation of SA-β-gal-positive cells ( n = 3 independent experiments, 6-8fields per experiment, 200–300 cells per field) ( f ), in control or CENEXIN1 -/- RCTE cells re-expressing CENEXIN1 or ODF2 (iso6) at day 10 after IR exposure. Scale bar, 100 μm. SA-β-gal staining ( g ), quantitation of SA-β-gal-positive cells ( n = 3 independent experiments, 3-4fields per experiment, 200–500 cells per field) ( h ), and relative mRNA level of SASP genes ( i ) in IL-1β-treated (3 ng/ml for 5 days) control or shCENEXIN1 RCTE cells. Scale bar, 50 μm. j Proposed working model: Exposure to irreparable stresses triggers the reorganization of microtubules (MTs), leading to the nucleation of sinc-MTs in the proximity of the nuclear envelop towards the ciliary base. Concurrently, the minus-end-directed kinesin KIFC3 is recruited to the ciliary base. It subsequently facilitates the transportation of the CENEXIN1-FBF1 cargo complex along the sinc-MTs, directing it towards the nucleus. This process initiates cellular senescence in stressed human cells. All results from n = 3 independent experiments. Data are the mean ± SEM. Statistical significance was determined using one-way ANOVA. Three experiments were repeated independently with similar results ( c ). Source data are provided as a file.

Article Snippet: Sub-cloning templates of human CENEXIN1 and KIFC3 were purchased from Addgene (#73334) and DNASU(#HsCD00442644), and ODF2(iso6) was kindly provided by Dr. Kyung Lee (NIH/NCI).

Techniques: Staining, Quantitation Assay, Control, Western Blot, Irradiation, Expressing

Journal: Nucleic Acids Research

Article Title: ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

doi: 10.1093/nar/gkaa815

Figure Lengend Snippet: ZFAT recruits BRD4 to centromeres. ( A ) Immunofluorescence images of ZFAT (HA) and BRD4 in HEK293 cells transiently expressing hZFAT-HA. The arrowhead indicates a transfected cell, as determined by the anti-HA antibody signal. ( B ) Immunofluorescence images of ZFAT, BRD4, CENP-A and DNA (DAPI) in HEK293 cells transiently expressing HA-hZFAT-EGFP. Enlarged images of the region enclosed by a white dotted line are shown. ( C ) Percentage of cells containing centromeric BRD4 foci in cells expressing EGFP or HA-hZFAT-EGFP, determined in (B). ( D ) ChIP-qPCR analysis of BRD4 at human repetitive DNA sequences using an anti-BRD4 antibody or control IgG in HEK293 cells expressing HA-hZFAT-EGFP or EGFP. The BRD4 protein levels are shown as the relative values to control IgG. (A, B) Scale bar, 5 μm. Data are representative of three independent experiments. (C, D) Data represent the mean ± SD of three independent experiments. * P < 0.05.

Article Snippet: The cDNA for human BRD4 (FHC11882) was purchased from Promega and cloned into an EGFP-C3 vector.

Techniques: Immunofluorescence, Expressing, Transfection

Journal: Nucleic Acids Research

Article Title: ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

doi: 10.1093/nar/gkaa815

Figure Lengend Snippet: ZFAT recruits BRD4 to centromeres through histone acetylation. ( A ) Schematic diagram of the BRD4 constructs used in (B). ( B ) Immunofluorescence images in HEK293 cells transiently expressing mCherry-hZFAT, and the full-length or deletion mutants of EGFP-BRD4. Enlarged images of the region enclosed by a white dotted line are shown. nls; nuclear localization signal. ( C ) Percentage of cells containing centromeric BRD4 foci in cells transfected with indicated expression vectors, determined in (B). ( D ) Immunofluorescence images of ZFAT (EGFP) and BRD4 in HEK293 cells expressing EGFP-hZFAT, treated with JQ1 or vehicle (Control). ( E ) Percentage of cells containing centromeric BRD4 foci in cells expressing EGFP-hZFAT, treated with JQ1 or vehicle, determined in (D). (B, D) Data are representative of three independent experiments. Scale bar, 5 μm. (C, E) Data represent the mean ± SD of three independent experiments.

Article Snippet: The cDNA for human BRD4 (FHC11882) was purchased from Promega and cloned into an EGFP-C3 vector.

Techniques: Construct, Immunofluorescence, Expressing, Transfection

Journal: Nucleic Acids Research

Article Title: ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

doi: 10.1093/nar/gkaa815

Figure Lengend Snippet: ZFAT recruits BRD4 to centromeres through KAT2B-mediated H4K8ac to promote the transcription of ncRNA. ( A ) ChIP-qPCR analysis of BRD4 at human repetitive DNA sequences using an anti-BRD4 antibody or control IgG in HEK293 cells transfected with indicated expression vectors and siRNAs. The BRD4 protein levels are shown as the relative values to control IgG. ( B ) Immunofluorescence images of ZFAT (HA) and BRD4 in HEK293 cells expressing the full-length or deletion mutants of hZFAT-HA. Enlarged images of the region enclosed by a white dotted line are shown. Scale bar, 5 μm. ( C ) Percentage of cells containing centromeric BRD4 foci in cells expressing the full length or deletion mutants of hZFAT-HA, determined in (B). ( D ) Immunoblotting analysis of ZFAT, KAT2B and BRD4 in HEK293 cells transfected with siRNAs for control or ZFAT (#1 and #5). ( E ) qRT-PCR analysis of centromeric ncRNA in HEK293 cells expressing EGFP or HA-hZFAT-EGFP (ZFAT), treated with JQ1 or vehicle. The RNA expression levels are shown as the relative values against those in vehicle-treated cells expressing HA-hZFAT-EGFP. ( F ) Immunoblotting analysis of ZFAT (HA) and BRD4 in HEK293 cells transfected with indicated expression vectors and siRNAs. N.S.; non-specific band. ( G ) qRT-PCR analysis of centromeric ncRNA in HEK293 cells transfected with indicated expression vectors and siRNAs. The RNA expression levels are shown as the relative values against those in cells transfected with control siRNA and HA-hZFAT-EGFP expression vector. ( H ) qRT-PCR analysis of centromeric ncRNA in HEK293 cells expressing EGFP or HA-hZFAT-EGFP (ZFAT), treated with α-amanitin or vehicle for 3 h. The RNA expression levels are shown as the relative values against those in vehicle-treated cells expressing HA-hZFAT-EGFP. (B, D, F) Data are representative of three independent experiments. (A, C, E, G, H) Data represent the mean ± SD of three independent experiments. * P < 0.05. (E, G, H) The RNA expression levels are normalized to those of β-actin.

Article Snippet: The cDNA for human BRD4 (FHC11882) was purchased from Promega and cloned into an EGFP-C3 vector.

Techniques: Transfection, Expressing, Immunofluorescence, Western Blot, Quantitative RT-PCR, RNA Expression, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

doi: 10.1093/nar/gkaa815

Figure Lengend Snippet: ZFAT is involved in chromosome segregation. ( A ) Immunofluorescence images of α-tubulin and DAPI in HT1080 cells transfected with indicated siRNAs. Data are representative of three independent experiments. Scale bar, 5 μm. ( B ) Percentage of cells with abnormal spindle morphology in mitotic cells transfected with indicated siRNAs, determined in (A). Data represent the mean ± SD of three independent experiments. ( C ) Model of ZFAT-regulated centromeric ncRNA transcription through the KAT2B–H4K8ac–BRD4 axis.

Article Snippet: The cDNA for human BRD4 (FHC11882) was purchased from Promega and cloned into an EGFP-C3 vector.

Techniques: Immunofluorescence, Transfection

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: PERK modulates the proximity and the mitochondrial Ca 2+ uptake at the EMCS but E-Syt1 does not. (A) Representative immunoblot for PERK, PSD, PSS1, PSS2 in shCTR and shPERK HeLa cells and quantification of PSD, PSS1, PSS2, and PERK, normalized on ACTIN (loading control) and relative to control condition (shCTR). The values shown are the mean ± SEM from three biological replicates analyzed using one sample t test. (B) Abundance of GFP-E-Syt1 pulled-down normalized on abundance of GFP-E-Syt1 in total lysate in HEK293-T cells transiently co-transfected with GFP-E-Syt1 and myc-tagged PERK full-length (FL) or myc-tagged PERK kinase dead mutant (PERK K618A ). The values plotted are the mean ± SEM from three biological replicates analyzed using unpaired Student’s t test. (C) Representative immunoblot for PERK, E-Syt1, CNX, and CYTC from total lysates, mito crude, mito pure, and MAMs fractions of PERK +/+ and PERK -/- MEFs cells. Arrows indicate E-Syt1, PERK, and a non-specific band. (D and E) Quantification of E-Syt1 levels at MAMs (D) and in the total lysate (E) normalized on CNX levels and relative to control condition (PERK +/+ ). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (F and G) Representative images (F) in situ PLA in shCTR and shPERK HeLa cells and quantification (G) of number of dots corresponding to IP3R3-VDAC1 interaction per nucleus and relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. Scale bar, 10 µm. (H and I) Representative images (H) in situ PLA in shCTR and shE-Syt1 HeLa cells and quantification (I) of number of dots corresponding to IP3R3-VDAC1 interaction per nucleus and relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. Scale bar, 10 µm. (J and K) Representative images (J) from EM analysis in shCTR and shE-Syt1 HeLa cells transfected with HRP-KDEL-myc and quantifications (K) of % mitochondria surface engaged into ER–mitochondria contact sites width below 30 nm (mitochondria occupancy %); mitochondrial average perimeter, number of mitochondria normalized on total mitochondrial perimeter and average number of mitochondria. The values plotted are the mean ± SEM ( n = 10 cells) analyzed using unpaired Student’s t test. Scale bar, 500 nm. (L and M) Representative traces (L) of mitochondrial calcium uptake after ER calcium depletion (ATP 100 µM) in shCTR and shPERK transiently transfected with mitochondrial Aequorin WT (mtAeqWT); quantification (M) of the mitochondrial peak of [Ca 2+ ]. The values plotted are the mean ± SEM from four biological replicates ( n = 22) analyzed using unpaired Student’s t test. Arrow indicates the addition of ATP. (N and O) Representative traces (N) of mitochondrial Ca 2+ uptake after ER calcium depletion (ATP 100 µM) in shCTR and shE-Syt1 transiently transfected with mtAeqWT; quantification (O) of the mitochondrial peak of [Ca 2+ ]. The values plotted are the mean ± SEM from three biological replicates ( n = 19 and n = 18 for shCTR and shE-Syt1, respectively) analyzed using unpaired Student’s t test. Arrow indicates the addition of ATP. *, P < 0.05; **, P <0.01; ***, P <0.001; and NS = not significant. Source data are available for this figure: .

Article Snippet: PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab ( ).

Techniques: Western Blot, Transfection, Mutagenesis, In Situ

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: PERK recruits the lipid transfer protein E-Syt1 at the EMCS. (A) Representative immunoblot for myc, GFP, and CNX after GFP pull-down showing PERK-E-Syt1 interaction in HEK293-T cells transiently co-transfected with myc-tagged PERK full-length (FL) or myc-tagged PERK kinase dead mutant (PERKK618A) and with eGFP-empty vector or eGFP-tagged E-Syt1. Untransfected cells are shown as negative control. In the eGFP-E-Syt1 transfected cells, a residual GFP signal is still visible as upper band (blue arrow) above PERK (red arrow) in the anti-myc panel. (B) Quantification of the PERK-E-Syt1 interaction normalized on GFP-pulled down and relative to control condition (PERK-myc). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (C) Representative immunoblot for eGFP and PERK showing the interaction of PERK and E-Syt1 in HEK293-T cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 pulled down. (D) Representative electron micrograph of ultrathin cryosections of HeLa cells transfected with eGFP-E-Syt1 and immunogold stained with anti-GFP (15 nm gold particles) and anti-PDI (10 nm gold particles). Black arrow denotes E-Syt1 detection at the sites of juxtaposition between the ER and the mitochondria membranes, while PDI, a general ER marker, remains in the ER lumen. Scale bar, 500 nm. (E) Relative quantification of the cellular distribution of E-Syt1 in the ER, EMCS (MAMs), and plasma membrane (PM). The values plotted are the mean ± SEM ( n = 16 cellular profiles). (F) Representative immunoblot for IP3R3, PERK, E-Syt1, CNX, VDAC1 and CYTC from total lysates, crude mitochondrial fraction (mito crude), purified mitochondrial fraction (mito pure) and MAM fraction of shCTR and shPERK HeLa cells. (G and H) Quantification of E-Syt1 level at MAMs (G) and total lysate (H) normalized on CNX levels and relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (I) Representative immunoblot for PERK in shCTR, shPERK, and shPERK + PERKK618A HeLa cells. ACTIN serves as loading control. (J) Representative images from eGFP-E-Syt1 transiently transfected and co-stained with MitoTracker Far Red in shCTR, shPERK and shPERK + PERKK618A HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (K) Colocalization analysis of E-Syt1 and MitoTracker Far Red in shCTR, shPERK and shPERK + PERKK618A HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates ( n = 26, n = 26, and n = 25 for shCTR, shPERK, and shPERK + PERKK618A respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (L) Abundance of PC, PI, PE from purified mitochondrial fractions of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: .

Article Snippet: PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab ( ).

Techniques: Western Blot, Transfection, Mutagenesis, Plasmid Preparation, Negative Control, Staining, Marker, Purification

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: E-Syt1 is found at the EMCS and regulates mitochondrial phospholipid abundance. (A) Representative immunoblot for IP3R3, E-Syt1, CNX, VDAC1, and CYTC from total lysates, mito crude, mito pure, and MAM fractions of shCTR and shE-Syt1 HeLa cells. (B) Abundance of PG from purified mitochondrial fractions of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from two biological replicates. (C) Abundance of PG, PC, PI, PE, PS from total cell lysates of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (D) Abundance of total mitochondrial phospholipids from purified mitochondrial fractions of shCTR and shE-Syt1 cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (E) Abundance of DG from purified mitochondrial fractions of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from two biological replicates. (F) Representative immunoblot for E-Syt1, PSD, PSS1, PSS2 in shCTR and shE-Syt1 HeLa cells and relative quantifications of PSD, PSS1, PSS2, and E-Syt1, normalized on ACTIN (loading control) and relative to control condition (shCTR). The values shown are the mean ± SEM from three biological replicates analyzed using one sample t test. (G) Mitotracker Green geometrical mean intensity (MFI) in shCTR and shE-Syt1 cells. The values plotted are the mean ± SEM from three biological replicates analyzed using unpaired Student’s t test. NS = not significant. Source data are available for this figure: .

Article Snippet: PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab ( ).

Techniques: Western Blot, Purification

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: PERK mediates E-Syt1 localization at the EMCS through E-Syt1 C2D-C2E domain. (A) Representative immunoblot for eGFP and PERK showing PERK-E-Syt1 interaction from mito crude fractions in DKO HeLa cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 pulled down. (B) Schematic representation of the E-Syt1 mutants anchored at the ER membrane used in this study; E-Syt1 full length (FL), E-Syt1-ΔSMP, E-Syt1-ΔDE, and E-Syt1-ΔCDE. (C) Representative immunoblot for eGFP and PERK showing PERK-E-Syt1 interaction from mito crude fractions in DKO HeLa cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1, E-Syt1-ΔCDE, E-Syt1-ΔSMP, and E-Syt1-ΔDE. (D–F) Quantification of PERK interaction normalized on E-Syt1-ΔCDE (D), E-Syt1-ΔSMP (E), or E-Syt1-ΔDE (F) eGFP pulled down and relative to control condition (eGFP-E-Syt1). The values plotted are the mean ± SEM from four biological replicates analyzed using one sample t test. (G) Representative images from eGFP-E-Syt1 full length, eGFP-E-Syt1-ΔDE or eGFP-E-Syt1-ΔSMP transiently transfected and co-stained with MitoTracker Far Red in DKO HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (H) Colocalization analysis of eGFP-E-Syt1/eGFP-E-Syt1-ΔDE/eGFP-E-Syt1-ΔSMP and MitoTracker Far Red in DKO HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates ( n = 24, n = 32, and n = 30 for eGFP-E-Syt1, eGFP-E-Syt1-ΔDE, and eGFP-E-Syt1-ΔSMP respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (I) Representative images from eGFP-E-Syt1 full length or eGFP-E-Syt1-ΔDE transiently co-transfected with STIM1-mCherry in DKO HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (J) Colocalization analysis of eGFP-E-Syt1/eGFP-E-Syt1-ΔDE and STIM1-mCherry in DKO HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates ( n = 28 and n = 25 for eGFP-E-Syt1 and eGFP-E-Syt1-ΔDE, respectively) analyzed using unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: .

Article Snippet: PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab ( ).

Techniques: Western Blot, Transfection, Plasmid Preparation, Staining

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: E-Syt1 interacts through its C2D-C2E domain with PERK. (A) Representative immunoblot for IP3R3, GFP, PERK, CNX, VDAC1, and CYTC from total lysate and mito crude fractions of DKO HeLa cells transiently transfected with eGFP-E-Syt1. (B and C) Representative immunoblot (B) for E-Syt1 and GFP in WT HeLa cells transiently transfected with eGFP and DKO HeLa cells transiently transfected with eGFP, eGFP-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, eGFP-E-Syt1-ΔCDE; quantification (C) of E-Syt1 expression levels normalized on ACTIN (loading control) in WT, DKO + E-Syt1, DKO + E-Syt1-ΔDE, DKO + E-Syt1-ΔSMP, or DKO + E-Syt1-ΔCDE HeLa cells. The values plotted are the mean ± SEM from three biological replicates using one-way ANOVA, with Tukey’s test for multiple comparisons. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 mutants transfected and E-Syt1 signals for E-Syt1 endogenous and eGFP-E-Syt1 mutants transfected. (D) Abundance of GFP pulled-down normalized on abundance of GFP in mito crude fraction in DKO HeLa cells transiently transfected with eGFP-tagged E-Syt1, E-Syt1-ΔCDE,E-Syt1-ΔSMP, and E-Syt1-ΔDE. The values plotted are the mean ± SEM from four biological replicates analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (E) Representative immunoblot for GFP and PERK showing PERK-E-Syt1 interaction from total lysate in HEK293-T cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1, E-Syt1-ΔSMP, E-Syt1-ΔDE, and E-Syt1-ΔCDE. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 mutants pulled down. (F) Abundance of GFP pulled-down normalized on abundance of GFP in total cell lysates in HEK293-T cells transiently transfected with eGFP-tagged E-Syt1, E-Syt1-ΔSMP, E-Syt1-ΔDE, and E-Syt1-ΔCDE. The values plotted are the mean ± SEM from biological replicates analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons . (G–I) Quantification of PERK interaction normalized on E-Syt1-ΔSMP (G), E-Syt1-ΔDE (H), or E-Syt1-ΔCDE (I) eGFP-pulled down and relative to control condition (eGFP-E-Syt1). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (J) Representative images from eGFP-E-Syt1 full length or eGFP-E-Syt1-ΔDE transiently co-transfected with STIM1-mCherry in PERK +/+ and PERK −/− MEFs cells. Scale bar, 10 µm. (K) Colocalization analysis of eGFP-E-Syt1/eGFP-E-Syt1-ΔDE and STIM1-mCherry in PERK +/+ and PERK −/− MEFs cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates ( n = 30, n = 29, n = 30, and n = 27 for PERK +/+ +E-Syt1-GFP, PERK +/+ +E-Syt1ΔDE, PERK −/− + E-Syt1-GFP, and PERK −/− + E-Syt1ΔDE, respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: .

Article Snippet: PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab ( ).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: The PERK-E-Syt1 complex transfers phospholipids at the EMCS. (A) Representative TLC image for converted NBD-PE and NBD-PS in WT, DKO and DKO cell transiently transfected with mCherry-E-Syt1 full length. Arrows indicate NBD-PS and converted NBD-PE heights. (B) Quantification of converted NBD-PE normalized on total NBD-PS + NBD-PE in WT, DKO and DKO + mCherry-E-Syt1 full length cells. The values plotted are the mean ± SEM from three biological replicates analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (C) Representative images of NBD-PS co-stained with MitoTracker Far Red in WT, DKO HeLa cells, and DKO HeLa cells transiently transfected with mCherry-E-Syt1 full length or mCherry-E-Syt1- ΔCDE. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (D) Colocalization analysis of NBD-PS and MitoTracker Far Red in WT, DKO, DKO + mCherry-E-Syt1 full length (FL), or DKO + mCherry-E-Syt1-ΔCDE HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates ( n = 21, n = 18, n = 18, and n = 17 for WT, DKO, DKO + mCherry-E-Syt1 FL, or DKO + mCherry-E-Syt1-ΔCDE, respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (E) Representative images of NBD-PS co-stained with MitoTracker Far Red in WT, DKO HeLa cells and DKO HeLa cells transiently transfected with mCherry-E-Syt1- ΔSMP. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (F) Colocalization analysis of NBD-PS and MitoTracker Far Red in WT, DKO, or DKO + mCherry-E-Syt1-ΔSMP HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates ( n = 29, n = 27, and n = 26 for WT, DKO, and DKO + mCherry-E-Syt1-ΔSMP respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: .

Article Snippet: PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab ( ).

Techniques: Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: PERK boosts the OCR independently of its ER stress activity but through E-Syt1 interaction. (A) Representative images showing the transfection efficiency (shown as merge of mCherry and BF channel) in WT and DKO HeLa cells transfected with mCherry empty vector (EV), DKO with mCherry-E-Syt1- ΔSMP and used in . Scale bar, 100 µm. (B) Representative images showing the transfection efficiency (shown as merge of mCherry and BF channel) in WT and DKO HeLa cells transfected with mCherry empty vector (EV), DKO with mCherry-E-Syt1, or mCherry-E-Syt1- ΔCDE and used in . Scale bar, 100 µm. (C) Representative immunoblot for E-Syt1 and GFP in WT HeLa cells electroporated with eGFP and DKO HeLa cells electroporated with eGFP, eGFP-E-Syt1, eGFP-E-Syt1-ΔSMP, eGFP-E-Syt1-ΔDE and used in . Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 mutants transfected and E-Syt1 signals for E-Syt1 endogenous and eGFP-E-Syt1 mutants transfected. (D) Quantification of E-Syt1 expression levels normalized on ACTIN (loading control) in WT, DKO + GFP-E-Syt1 full length, DKO + GFP-E-Syt1-ΔSMP, and DKO + GFP-E-Syt1-ΔDE HeLa cells. The values plotted are the mean ± SEM from three biological replicates analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. NS = not significant. Source data are available for this figure: .

Article Snippet: PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab ( ).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

doi: 10.1083/jcb.202206008

Figure Lengend Snippet: The PERK-E-Syt1 axis maintains mitochondrial respiration. (A and B) OCR of shCTR, shPERK, and shPERK + PERKK618A HeLa cells in galactose media (A); quantification of basal respiration, ATP production and maximal respiration (B). The values plotted are the mean ± SEM from four biological replicates using one-way ANOVA, with Tukey’s test for multiple comparisons. (C and D) OCR of CTR and p.W681X PERK mutant human fibroblasts in galactose media in untreated conditions and after 2 h pre-treatment with PERK inhibitor (PKI) GSK2606414 1 µM (C); quantification of basal respiration, ATP production and maximal respiration (D). The values plotted are the mean ± SEM from three biological replicates using one-way ANOVA, with Tukey’s test for multiple comparisons. (E) Representative immunoblot for PERK and PERK substrate eIf2α (p-eIF2α and total (Tot) EIF2α) in CTR human fibroblasts, respectively in untreated conditions, treated with PERK inhibitor (PKI) GSK2606414 1 µM, ER-stress inducer Thapsigargin (TG) 2 µM for 2 h and TG 2 h + PKI. ACTIN serves as loading control. (F and G) OCR of WT HeLa cells electroporated with eGFP, DKO HeLa cells electroporated with eGFP, eGFP-E-Syt1, eGFP-E-Syt1-ΔDE, or eGFP-E-Syt1-ΔSMP in galactose media (E); quantification of basal respiration, ATP production, and maximal respiration (F). The values plotted are the mean ± SEM from five biological replicates using one-way ANOVA, with Tukey’s test for multiple comparisons. (H) Schematic representation of the effects of E-Syt1 full length (FL), E-Syt1-ΔSMP, and E-Syt1-ΔDE on mitochondrial metabolism at the EMCS. *, P < 0.05; **, P < 0.01; ***,P < 0.001; and NS = not significant. Source data are available for this figure: .

Article Snippet: PERK.K622A.9E10.pCDNA myc tagged (21815; Addgene plasmid), PERK.WT.9E10.pCDNA myc tagged (21814; Addgene plasmid), eGFP-E-Syt1 (66830; Addgene plasmid) p-eGFP-C3 empty vector plasmid (2489; Addgene plasmid) and pmCherry-C1 empty vector plasmid (3552; Addgene plasmid) were obtained from Addgene. mCherry-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, and mCherry-E-Syt1-ΔCDE were a kind gift from Pietro De Camilli lab ( ).

Techniques: Mutagenesis, Western Blot