Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PtdIns(4,5)P 2 interacts with NRF2 in the nucleus. A , endogenous NRF2 was IPed from MDA-MB-231 cells, and PtdIns(4,5)P 2 was detected by fluorescent IB. IgG was used as a negative control. B , MDA-MB-231 cells were cultured from low confluency in media containing [ 3 H] myo -inositol or unlabeled myo -inositol. After 72 h, cells were treated with 200 μM DEM or vehicle for 4 h before being processed for IP against NRF2. NRF2 and PtdIns(4,5)P 2 were confirmed and quantified ( A ) by fluorescent WB and ImageJ before samples were resolved by SDS-PAGE and the gel sectioned corresponding to the NRF2 immunoblot. Gel sections from the indicated lanes were then dissolved and analyzed by liquid scintillation counting (LSC). Graph is mean ± SD C , HEK293FT cells were transiently transfected with FLAG-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. D and E , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( D ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( E ) is shown as mean ± SD of three independent experiments with n = 10 cells scored in each independent experiment. F and G , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( F ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( G ) is shown as mean ± SD of three independent experiments with n = 15 cells scored in each independent experiment. For all panels , n = 3 independent experiments; scale bar represents 5 μm. DAPI, 4',6-diamidino-2-phenylindole; DEM, diethyl maleate; HEK293FT, human embryonic kidney 293FT cell line; IB, immunoblotting; IF, immunofluorescence; IgG, immunoglobulin G; IP, immunoprecipitation; IPed, immunoprecipitated; NRF2, nuclear factor erythroid 2–related factor 2; PLA, proximity ligation assay; PtdIns(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; WB, Western blot.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Negative Control, Cell Culture, SDS Page, Western Blot, Transfection, Staining, Microscopy, Immunofluorescence, Immunoprecipitation, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PIPKIγ controls NRF2 stability. A and B , the expression of NRF2 was analyzed by IB in MDA-MB-231 cells with deletion of PIPKIα, PIPKIγ, PIPKIIα, or PIPKIIβ ( A ). The intensity of the immunoblots was quantified, and the graph is shown ( B ) as mean ± SD. C , KD of PIPKIγ using two shRNAs in HCT116 cells. Cells were treated with vehicle or 100 μM tBHQ for 4 h. The expression of NRF2 was analyzed by IB, and the graph is shown as mean ± SD. D , MDA-MB-231 cells (parental or with PIPKIγ deletion) were treated with 100 μM tBHQ for 4 h. NRF2 protein expression was analyzed by IB. E , MDA-MB-231 cells transfected with control siRNAs or siRNAs targeting PIPKIγ were treated with or without 10 μM MG132 for the indicated time. NRF2 protein levels were determined by IB and quantified ( B ) by ImageJ. For all panels , n = 3 independent experiments. KD, knockdown; IB, immunoblotting; NRF2, nuclear factor erythroid 2–related factor 2; PIPKIγ, phosphatidylinositol phosphate kinase γ; tBHQ, tertiary butylhydroquinone.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Expressing, Western Blot, Transfection, Control, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PIPKIγ binds nuclear NRF2 . A and B , MDA-MB-231 ( A ) or HCT116 ( B ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB and quantified by ImageJ. C and D , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( C ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( D ) is shown as mean ± SD of three independent experiments with n = 10 cells scored in each independent experiment. E and F , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( E ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( F ) is shown as mean ± SD of three independent experiments with n = 15 cells scored in each independent experiment. G and H , MDA-MB-231 cells with or without PIPKIγ knockdown (KD) were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( G ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LASX (Leica). The experiments were repeated three times. The graph is shown as mean ± SD of n = 15 cells from one representative experiment ( H ). For all panels , n = 3 independent experiments; scale bar represents 5 μm. DAPI, 4',6-diamidino-2-phenylindole; DEM, diethyl maleate; IB, immunoblotting; IF, immunofluorescence; IPed, immunoprecipitated; NRF2, nuclear factor erythroid 2-related factor 2; PIPKIγ, phosphatidylinositol phosphate kinase γ; PLA, proximity ligation assay.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Staining, Microscopy, Knockdown, Western Blot, Immunofluorescence, Immunoprecipitation, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: HSP27 interacts with NRF2 . A and B , endogenous NRF2 was IPed from MDA-MB-231 ( A ) or MCF7 ( B ) cells, and HSP27, αB-crystallin, HSP40, HSP60, and HSP90 were analyzed by IB. IgG was used as a negative control. C , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous NRF2 was IPed, and HSP27 was analyzed by IB. D , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous HSP27 was IPed, and NRF2 was analyzed by IB. E , quantification using ImageJ of co-IPed protein levels ( C and D ). F and G , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF staining against HSP27 and NRF2 ( F ). DAPI was used to stain nuclei. The images ( F ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The experiments were repeated three times, and the graph ( G ) is shown as mean ± SD of three independent experiments with n = 10 cells scored in each independent experiment. H and I , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between HSP27 and NRF2. DAPI was used to stain nuclei. The images ( H ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( I ) is shown as mean ± SD of three independent experiments with n = 15 cells scored in each independent experiment. For all panels , n = 3 independent experiments; scale bar represents 5 μm. DAPI, 4',6-diamidino-2-phenylindole; DEM, diethyl maleate; HSP27, heat shock protein 27; IB, immunoblotting; IF, immunofluorescence; IgG, immunoglobulin G; IPed, immunoprecipitated; NRF2, nuclear factor erythroid 2-related factor 2; PLA, proximity ligation assay.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Negative Control, Staining, Microscopy, Western Blot, Immunofluorescence, Immunoprecipitation, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PtdIns(4,5)P 2 binding to NRF2 promotes its interaction with sHSPs. A , schematic representation of NRF2 truncation mutants used in the study. B , recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB and the bound NRF2 mutants were quantified for HSP27 ( C ) and αB-crystallin ( D ) using ImageJ. E , recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. F and G , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 ( F ) or αB-crystallin ( G ) was analyzed by IB. For all panels , n = 3 independent experiments. HSP27, heat shock protein 27; PtdIns(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; sHSP, small heat shock protein.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Binding Assay, Recombinant, Mutagenesis, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: HSP27 stabilizes NRF2. A , MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27, and NRF2 expression was analyzed by IB 72 h later. The graph is shown as mean ± SD. B , RT–PCR analysis of NFE2L2 mRNA in MDA-MB-231 cells. NFE2L2 mRNA levels were normalized to GAPDH mRNA. The graph is shown as mean ± SD of n = 4 independent experiments. C , MDA-MB-231 cells transfected with control siRNAs or siRNAs targeting HSP27 were treated with or without 10 μM MG132 for the indicated time. NRF2 protein levels were determined by IB. D and E , HCT116 ( D ) or HS578 ( E ) cells were transiently transfected with control siRNAs or siRNAs targeting HSP27. About 68 h later, transfected cells were treated with vehicle or 100 μM tBHQ for an additional 4 h, and NRF2 expression was then analyzed by IB. The graph is shown as mean ± SD. F , A549 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27, and NRF2 expression was analyzed by IB 72 h later. The graph is shown as mean ± SD. G , 0.5 μg of recombinant His-tagged NRF2 and 9 μg KEAP1 were incubated with the indicated concentrations of HSP27. NRF2 was pulled down, and the bound KEAP1 and HSP27 were analyzed by IB. For all panels , n = 3 independent experiments unless otherwise indicated. HSP27, heat shock protein 27; IB, immunoblotting; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2–related factor 2; tBHQ, tertiary butylhydroquinone.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Transfection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Recombinant, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PIPKI γ and HSP27 regulate HO-1 expression, ROS levels, and oxidative stress–induced cell death. A , HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 was analyzed by IB and quantified by ImageJ ( C ). B , MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. C and D , ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( C ) or PIPKIγ ( D ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± SD. E and F , cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( E ) or PIPKIγ ( F ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± SD. G , a schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 , and sHSPs. For all panels , n = 3 independent experiments. DEM, diethyl maleate; HO-1, heme oxygenase-1; HSP27, heat shock protein 27; IB, immunoblotting; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NRF2, nuclear factor erythroid 2–related factor 2; PIPKIγ, phosphatidylinositol phosphate kinase γ; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; ROS, reactive oxygen species; sHSP, small heat shock protein.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Expressing, Control, shRNA, Transfection, MTT Assay, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Splicing factor Sf3b1 facilitates maintenance of neuronal dendrites by modulating mitochondrial health

doi: 10.1007/s00018-025-05860-0

Figure Lengend Snippet: Reduced growth of dendritic arbors upon depletion of Sf3b1 in ddaC and ddaF neurons. ( A ) Schematics showing three DrosDel lines, Df(2L)ED94 , Df(2L)ED49, and Df(2L)ED62 , used for lethality mapping in which deleted regions are filled with red dots, the genomic locations of Sf3b1 and lectin-24Db in the chromosomal 2L region are labeled by black boxes, transcripts are represented by blue bars, exons are represented by yellow bars, and untranslated regions are shown by gray bars, with arrows indicating the direction of transcription. The lectin-24Db MI11191 line carries a MiMIC insertion (black diamond) at the lectin-24Db locus and a deleted region (black dotted) encompassing Sf3b1 . The newly isolated Sf3b1 mt allele is also shown. ( B-E ) Images display MARCM clones of ddaC neurons for the ( B ) control ( GAL4 5-40 UAS-Venus::pm SOP-FLP #42 ; Tub-GAL80 FRT40A/FRT40A ), ( C ) Sf3b1 mt mutant, ( D ) Sf3b1 mt mutant carrying UAS-Sf3b1, and ( E ) Sf3b1 mt mutant carrying UAS-SF3B1 Lines. Scale bars are 50 μm. ( F ) Quantification of dendritic arbor terminals. ( G-I ) Images show MARCM clones of ddaF neurons for the ( G ) control, ( H ) Sf3b1 mt mutant, and ( I ) Sf3b1 mt mutant carrying UAS-Sf3b1 lines . Boxed regions are magnified underneath to show spike protrusions. Scale bars are 50 μm. ( J ) Quantification of dendritic arbor terminals. In ( F, J ), bar graphs represent the mean ± SEM, and comparisons between data groups are indicated by lines with statistical significance tested by Student’s t -test and shown as **** for p < 0.0001

Article Snippet: SF3B1 plasmids , Addgene [ ] , # 82576.

Techniques: Labeling, Isolation, Clone Assay, Control, Mutagenesis

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Splicing factor Sf3b1 facilitates maintenance of neuronal dendrites by modulating mitochondrial health

doi: 10.1007/s00018-025-05860-0

Figure Lengend Snippet: Retracted branch growth in Sf3b1 knockdown neurons at the late larval stage. ( A ) Images display ddaC neurons from ppk-GAL4 -driven lacZ control and Sf3b1 RNAi transgenic larvae at 72, 90 and 120 h AEL, with dendrites marked by CD4-tdTom. Scale bars are 100 μm. ( B ) Quantification of dendritic terminal numbers. ( C ) Images of ddaC dendrites were acquired at 105 h AEL and 3 h later for the lacZ control and Sf3b1 RNAi knockdown lines. Changes in the length of terminal branches have been color-coded: newly emerged (red); vanished (blue); extended (pink); and retracted (cyan), all within the 3-h period. ( D ) Percentages of terminal branches exhibiting different dynamics (emerge, vanish, extend, and retract). In ( B, D ), bar ( B ) and dot ( D ) graphs show the mean ± SEM, and comparisons between two data groups were processed by Student’s t -test with ** for p < 0.01, *** for p < 0.001, **** for p < 0.0001, and ns for no significance ( p > 0.05)

Article Snippet: SF3B1 plasmids , Addgene [ ] , # 82576.

Techniques: Knockdown, Control, Transgenic Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Splicing factor Sf3b1 facilitates maintenance of neuronal dendrites by modulating mitochondrial health

doi: 10.1007/s00018-025-05860-0

Figure Lengend Snippet: Altered mRNA splicing events in Sf3b RNAi knockdown larvae. ( A ) Schematic showing genetic crosses of da-GAL4 females with w 1118 (control) or Sf3b RNAi males to produce larval offspring 24 h AEL for RNA sequencing and analyses. ( B ) The pie chart shows the result of rMATS analysis, with a threshold of |Δψ|> 0.1 and FDR ≤ 0.05, that identified 2,228 altered splicing events across 1,118 genes for the alternative 3’ splicing site (A3SS), alternative 5’ splicing site (A5SS), retained intron (RI), skipped-exon (SE), and mutually exclusive exon (MXE) categories in Sf3b1 RNAi knockdown larvae. Numbers of events (n) are displayed. The GO enrichment analysis of alternatively spliced genes identified from rMATS is shown at right. ( C ) Venn diagram depicting the 185 genes displaying intron splicing variants, 45 of which (3.6%) overlap with the 1,118 genes identified by rMATS. The GO enrichment analysis of genes exhibiting intron splicing variation, as derived by aligning CIGAR strings to the reference genome, is presented at right. The GO terms have been grouped into Molecular Function (MF), Cellular Component (CC), and Biological Process (BP). Circle size represents gene count, with darker colors indicating lower adjusted p-values (adj.p), as calculated using the Benjamini–Hochberg method, and denoting greater significance

Article Snippet: SF3B1 plasmids , Addgene [ ] , # 82576.

Techniques: Knockdown, Control, RNA Sequencing, Derivative Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Splicing factor Sf3b1 facilitates maintenance of neuronal dendrites by modulating mitochondrial health

doi: 10.1007/s00018-025-05860-0

Figure Lengend Snippet: Validation of altered mRNA splicing following Sf3b1 knockdown. ( A-D ) Integrative Genomics Viewer (IGV) was used to display base coverage in da-GAL4 control (red) and Sf3b1 RNAi knockdown (blue) conditions, with counts within circles indicating splice junction reads and the y-axis showing maximum exon coverage. ( A ) SE events for Sf3b3 and Opa1 . ( B ) RI events for TFAM and prel . ( C ) A5SS events for lap and Gptx . ( D ) A3SS events for hlk and msn . Gene schematics show the direction of transcription (black arrows), exons as blocks, introns as lines, and alternatively used coding regions in pale green. Exon numbers have been named according to the RA transcripts of Opa1 , TFAM , Gptx , hlk , and msn , and the RC transcript of the remaining genes. Gel images show amplified cDNA for control and Sf3b1 RNAi by RT-PCR with primers marked by red arrows and schematics for predicted splicing events at right. In addition to alternative splicing, other splicing variants in prel and msn were detected, including an unannotated retained intron in prel and an extra PCR product for msn (*)

Article Snippet: SF3B1 plasmids , Addgene [ ] , # 82576.

Techniques: Biomarker Discovery, Knockdown, Control, Amplification, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Splicing factor Sf3b1 facilitates maintenance of neuronal dendrites by modulating mitochondrial health

doi: 10.1007/s00018-025-05860-0

Figure Lengend Snippet: Genes downregulated upon Sf3b1 RNAi knockdown are associated with mitochondrial functions. ( A, B ) GO enrichment analysis for significantly upregulated ( A ) or downregulated ( B ) DEGs following Sf3b1 RNAi knockdown. The GO terms have been categorized as Molecular Function (MF), Cellular Component (CC), or Biological Process (BP). Circle size reflects gene counts, with darker colors indicating lower adjusted p-values (adj.p) and denoting greater significance. ( C ) Volcano plot displaying significantly upregulated (log 2 FC > 1, red) and downregulated (log 2 FC < −1, blue) genes, both with [FDR] < 0.05. Those associated with mitochondrial functions, identified using the MitoMax database, are marked with darker dots, revealing a significant presence in the downregulated category (dark blue). ( D ) PANGEA network of enriched functional terms. The nodes represent enriched terms, emphasizing energy-associated processes among the downregulated mitochondrial-related genes highlighted in dark blue in ( C ). These include a."Oxidation–reduction process"(GO:0055114), b."Mitochondrial respiratory chain complex assembly"(GO:0033108), c."Electron transport chain"(GO:0022900), d."Mitochondrial translation"(GO:0032543), e."Fatty acid oxidation"(GO:0019395), f."Citrate cycle (TCA cycle)"(KEGG: map00020), g."Oxidative phosphorylation"(KEGG: map00190), and h."Complex I biogenesis"(Reactome: R-DME-6799198)

Article Snippet: SF3B1 plasmids , Addgene [ ] , # 82576.

Techniques: Knockdown, Functional Assay, Phospho-proteomics

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Splicing factor Sf3b1 facilitates maintenance of neuronal dendrites by modulating mitochondrial health

doi: 10.1007/s00018-025-05860-0

Figure Lengend Snippet: Mitochondrial defects in Sf3b1 knockdown C4da neurons. ( A ) Images show Dendra2-labeled mitochondria in the cell bodies of C4da neurons from lacZ control and Sf3b1 RNAi knockdown lines driven by ppk-GAL4 , with enlarged views of yellow boxed regions at right. Scale bars are 5 μm. ( B ) Averaged mitochondrial length and area of neuronal cell bodies. ( C ) Images show the distribution of mitochondria in C4da neurons of lacZ control and Sf3b1 RNAi knockdown larvae at 120 h AEL, with Dendra2 labeling mitochondria and CD4-td-Tom labeling dendrites. Scale bars are 100 μm. Magnified views of selected boxed areas, showing combined images and individual images of dendrites (red) and mitochondria (green). ( D ) Quantification of mitochondrial density within major dendritic processes at 72, 90, and 120 h AEL. ( E ) Representative pseudo-color images (rainbow LUT, scale 0–1.6) illustrate ATP sensor intensity in lacZ control and Sf3b1 RNAi neurons following 1 mM Antimycin A treatment for 0, 5, 10, 20, and 30 min. Scale bar represents 10μm. ( F ) Line graph shows the iATPSnFR 1.0 :mRuby3 ratios in lacZ control (blue) and Sf3b1 RNAi (yellow) neurons following 1 mM Antimycin A treatment for 0, 5, 10, 20, and 30 min. The open circle represents C4da neuron ATP level in larvae dissected without Antimycin A treatment, incubated in HL3 medium for 60 min. Data represent mean ± SEM. Statistical significance between control and experimental groups at each indicated time point was determined using Student's t-test , * for p < 0.05 and ns, not significant. ( G ) Images show dendrites outlined by mRuby3 signals of the mRuby3-iATPSnFR 1.0 sensor driven by ppk-GAL4 in lacZ control and Sf3b1 RNAi knockdown larvae at 120 h AEL. Panels at right display the straightened dendritic processes (yellow lines in images at left) with images of mRuby3 (red), iATPSnFR 1.0 (green), and the iATPSnFR 1.0 :mRuby3 ratio pseudo-colored using a rainbow lookup table from 0–1. ( H ) Quantification of the iATPSnFR 1.0 :mRuby3 ratio. The bar graph shows the ratios for control and Sf3b1 RNAi dendrites at 72, 90, and 120 h AEL. Each data point represents the ratio from an individual segment mask. ( B, D, H ) Bar graphs show means ± SEM, with *** for p < 0.001, **** for p < 0.0001, and ns for no significance

Article Snippet: SF3B1 plasmids , Addgene [ ] , # 82576.

Techniques: Knockdown, Labeling, Control, Incubation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Splicing factor Sf3b1 facilitates maintenance of neuronal dendrites by modulating mitochondrial health

doi: 10.1007/s00018-025-05860-0

Figure Lengend Snippet: Rescue of impaired dendritic branching and diminished mitochondrial densities in Sf3b1 RNAi knockdown neurons. ( A ) Images display ppk-GAL4 -driven CD4-tdTom-labeled C4da neurons with Sf3b1 RNAi knockdown and co-expression of lacZ , AMPKα CA , Buffy , Lim3 , Pink1 , park , Nmnat , Marf , or PPP1R15 , Scale bars are 100 μm. ( B ) Quantification of terminal points in Sf3b1 RNAi knockdown neurons co-expressing one of the genes in (A) . ( C ) Mitochondrial distribution in Sf3b1 RNAi knockdown C4da neurons, as visualized using Dendra2 labeling for mitochondria and CD4-td-Tom for dendrites. Images show the effects of overexpressing lacZ , AMPKα CA , Pink1 , park , or Nmnat , driven by ppk-GAL4 . Scale bars are 100 μm. Enlarged boxed areas on right show merged and separate dendrite (red) and mitochondria (green) images. ( D ) Quantification of mitochondrial density in dendritic processes. In ( B, D ), bar graphs represent mean ± SEM. Statistical significance was determined relative to the lacZ control by Student’s t -test and is shown as *** for p < 0.001, **** for p < 0.0001, and ns for no significance

Article Snippet: SF3B1 plasmids , Addgene [ ] , # 82576.

Techniques: Knockdown, Labeling, Expressing, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Splicing factor Sf3b1 facilitates maintenance of neuronal dendrites by modulating mitochondrial health

doi: 10.1007/s00018-025-05860-0

Figure Lengend Snippet: Mitochondrial turnover in Sf3b1 RNAi neurons is partially restored by AMPKα CA , Pink1 , or park overexpression. ( A ) Images show merged green and red channels of MitoTimer-labeled mitochondria in the soma and dendritic processes (arrowheads mark the mitochondria in dendrites) in C4da neurons of lacZ control larvae, as well as for Sf3b1 RNAi larvae overexpressing lacZ , AMPKα CA , Pink1, or park . The bottom panel shows a pseudo-color representation of the red:green ratio ranging from 0–1. Scale bars are 5 μm. ( B ) Quantification of MitoTimer fluorescence ratios in the cell bodies (grey bars) and dendrites (pink bars) of C4da neurons. Each dot in the bar graph represents the red:green ratio for an individual mitochondrial ROI. Red/green ratios are expressed as percentages (raw ratio × 100). ( C ) Quantification of numbers of red puncta in the cell body. In ( B, C ), means ± SEM are shown. Statistical significance was determined by Student’s t -test, with comparisons between the indicated groups. Significance levels are denoted as * for p < 0.05, ** for p < 0.01, **** for p < 0.0001, and ns for no significance

Article Snippet: SF3B1 plasmids , Addgene [ ] , # 82576.

Techniques: Over Expression, Labeling, Control, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: PEX1 G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome

doi: 10.1016/j.jbc.2025.108467

Figure Lengend Snippet: PEX1 G843D degradation is hindered by fusion to a deubiquitinase OTUB1 . A , ubiquitin ligase related proteins identified by anti-FLAG copurification MS/MS in FLAG-PEX1 WT , FLAG-PEX1 G843D , PEX1 WT -FLAG, and PEX1 G843D -FLAG cell lines. The plot shows the comparison between WT and G843D samples on a subset of total identified proteins related to ubiquitin interacting proteins. Comparisons are the mean of total precursor intensity from two WT and two G843D samples . B , representative immunoblots and quantification of PEX1 levels of PEX1 G843D/G843D HCT116 CRISPRi (dCas9-KRAB) cell lines with knockdown of UBR5 and UBE2O candidates identified from the IP-MS experiment (PEX1 = 142 kDa, SF = Stain-Free total protein). Knockdown of mRNA transcripts verified in . Cells were treated with cycloheximide at T = 0 and samples collected after 24 h. Mean ± SD is shown from N = 3 biological replicates for each timepoint. C , representative immunoblots of PEX1-OTUB1 fusions in a CHX-chase assay. OTUB1 C91S is catalytically inactive. D , quantification of replicate experiments similar to ( C ). Mean ± SD is shown from N = 3 biological replicates for each time point. CHX, cycloheximide; IP-MS, immunoprecipitation mass-spectrometry.

Article Snippet: Transient transfection plasmids containing the PEX1 G843D -OTUB1 fusions were cloned by Gibson assembly to insert PEX1 G843D into the transient transfection OTUB1 or OTUB1 C91S pcDNA3.1 plasmids (Addgene #118209 and Addgene #118210, respectively).

Techniques: Ubiquitin Proteomics, Copurification, Tandem Mass Spectroscopy, Comparison, Western Blot, Knockdown, Protein-Protein interactions, Staining, Immunoprecipitation, Mass Spectrometry