Journal: International Journal of Biological Sciences

Article Title: The Interaction of CircESR1 and HNRNPAB Regulates Cell Cycle Transition of Breast Cancer Cell

doi: 10.7150/ijbs.126014

Figure Lengend Snippet: Estrogen promotes HNRNPAB expression via SP1. (A) Immunoblot assessment of HNRNPAB in 2 human normal breast epithelial cell lines and 11 human BC cell lines. (B) CPTAC database analyzed the relative HNRNPAB and ERα protein level in 105 BC patients. P value was determined by Pearson correlation analysis. (C) The relative expression of TFF1 and HNRNPAB mRNAs after stimulated with 10 nM E 2 in estrogen-deprived MCF-7 cells for 48 h analyzed by qRT-PCR. (D) Immunoblot assessment of HNRNPAB expression in MCF-7 with or without 10 nM E 2 in estrogen-deprived MCF-7 cells for 72 h, or in short-term oestrogen deprivation (STED) MCF-7 cells for 7 days and parental cells. (E) The schematic of HNRNPAB promoter region sequence selected from the transcription start site of -2 kb~+100 bp. (F) MCF-7 cells were transfected with pGL3 basic reporter vector containing HNRNPAB promoter, and pRL-TK reporter control vector containing luciferase activity, as well as control siRNA or different siRNA sequences of JUN , FOS , FOXA1 and SP1 . The relative fluorescence activity was measured. (G) CPTAC database analyzed the relative HNRNPAB and SP1 protein level in 68 ER+ BC patients. P value was determined by Pearson correlation analysis. (H) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector containing different regions of HNRNPAB promoter. The relative fluorescence activity was measured. (I) The schematic of three predicted conserved SP1 binding sites on the upstream -425 bp~+100 bp regions of the human HNRNPAB gene. Red boxes represent the predicted SP1 binding sites. (J) The DNA regions enriched by SP1 in ChIP assay were verified by PCR. (K) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector or containing “b” or “b” mutant regions. The relative fluorescence activity was measured. (L) The relative expression of SP1 and HNRNPAB mRNAs in MCF-7 cells bearing control or SP1 shRNAs analyzed by qRT-PCR. (M) Immunoblot assessment of SP1 and HNRNPAB expression in MCF-7 cells with or without SP1 shRNAs in the presence or absence of 10 nM E 2 for 48 h. Data was shown as mean ± S.D. from three independent experiments. Unpaired two-tailed Student's t test (C, H, K) and one-way ANOVA followed by Tukey's multiple comparisons test (F, L). ns, P >0.05; ***, P <0.001; ****, P <0.0001.

Article Snippet: For transcription factor mediated HNRNPAB expression, 0.2 μg pGL3 Basic luciferase reporter (RRID: Addgene_48743), 50 nmol JUN or FOS or FOXA1 or SP1 siRNAs and 0.02 μg pRL-TK plasmid (RRID: Addgene_11313) were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Sequencing, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Fluorescence, Binding Assay, Mutagenesis, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF interacts with eIF4E at the m 7 G cap, independent of intact eIF4G1. ( a ) Reporter assay quantifying the relative Renilla luciferase expression from the indicated 3′ UTR luciferase reporters in untreated and TGF-β-treated MCF-10A cells. Data were normalized to Firefly luciferase expression and are presented as fold change of this normalized signal relative to CXCR4 reporter in untreated MCF-10A cells. ( b ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of indicated Renilla luciferase reporters (pRL-TK) in untreated and TGF-β-treated MCF-10A cells. Data were normalized to endogenous ACTB expression. ( c ) As in (a), with the indicated 3′ UTR luciferase reporters driven from an EMCV internal ribosomal entry site (IRES) in untreated and TGF-β-treated MCF-10A cells. ( d ) As in (b), for reporter assays in panel (c). ( e ) Right six lanes—immunoblots of indicated immunoprecipitates from whole-cell lysates derived from MCF-10A cells treated with TGF-β for 72 h. One half of each total immunoprecipitate was digested with RNase A prior to immunoblotting with the indicated antibodies. Right six lanes—as in the left six lanes, but lysates were digested with coxsackievirus 2A protease to cleave eIF4G1 before immunoprecipitation. CT = C-terminal; FL = full length; NT = N-terminal. ( f ) m 7 GTP cap analog binding assays utilizing cytosolic extracts derived from MCF-10A cells treated with TGF-β for 72 h. As above, one half of each extract was digested with coxsackievirus 2A protease to cleave eIF4G1 before the assay. ( g ) Proximity ligation assays using the indicated pairs of antibodies on MCF-10A cells treated with TGF-β for 72 h. In all panels, results are representative of at least three independent experiments and error bars depict mean ± standard deviation (SD) of aggregate replicates performed in triplicate. NS: not significant; * P -value < 0.05 (Student’s t-test).

Article Snippet: WT or GRE deletion mutant CRLF1 and SNAI1 3′ UTRs were fused downstream of the Renilla luciferase coding sequence in the broadly used pRL-TK CXCR4 6x reporter plasmid ([ ], Plasmid #11 308, Addgene, Cambridge, MA) as described before [ ].

Techniques: Reporter Assay, Luciferase, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Derivative Assay, Immunoprecipitation, Binding Assay, Ligation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF1 stimulates translation of GRE-containing EMT effector mRNAs in the context of reduced eIF4G1 function. ( a ) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs ( EGR3, FOSB, JUNB, SNAI1 ) from TGF-β-treated MCF-10A cells using anti-CELF1, anti-eIF4E, and anti-eIF4G1 antibodies or mouse and rabbit IgG. ACTB and GAPDH are non-GRE-containing negative control mRNAs. ( b ) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs ( EGR3, FOSB, JUNB, SNAI1 ) from TGF-β-treated MCF-10A cells using tandem anti-eIF4E/anti-CELF1 immunoprecipitation, tandem anti-eIF4E/anti-eIF4G1 immunoprecipitation, or tandem immunoprecipitation with mouse and rabbit IgGs. ACTB and GAPDH are non-GRE-containing negative controls. ( c, d ) Efficiency of in vitro translation of indicated capped and polyadenylated Renilla luciferase reporter mRNAs in mock or 2A protease-digested cell-free extract. ( e, f ) Efficiency of in vitro translation of reporter mRNAs as described in panels (c) and (d), but with mock-depleted ( Beads ) cell-free extract, eIF4G1-immunodepleted ( ID ) cell-free extract, or eIF4G1-immunodepleted cell-free extract reconstituted by addition of 20 nM enriched eIF4G1 and/or an equivalent concentration of recombinant CELF1. In panels (c–f), all extracts were derived from TGF-β-treated MCF-10A cells transiently transfected with shRNAs targeting either GLB1 (c, e) or CELF1 (d, f). CXCR4 = control, WT = wild-type 3′ UTR, ΔGRE =3′ UTR with deletion of GRE. In all panels, results are representative of at least three independent experiments. Error bars depict mean ± standard deviation (SD) of aggregate replicates performed in triplicate. NS: not significant; * P -value < 0.05 ( a, b, e, f : ANOVA with Dunnet’s post-hoc test; c, d : Student’s t-test).

Article Snippet: WT or GRE deletion mutant CRLF1 and SNAI1 3′ UTRs were fused downstream of the Renilla luciferase coding sequence in the broadly used pRL-TK CXCR4 6x reporter plasmid ([ ], Plasmid #11 308, Addgene, Cambridge, MA) as described before [ ].

Techniques: Cross-linking Immunoprecipitation, Quantitative RT-PCR, Negative Control, Immunoprecipitation, In Vitro, Luciferase, Concentration Assay, Recombinant, Derivative Assay, Transfection, Control, Standard Deviation

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) AlphaFold prediction of full-length BLTP3A with ConSurf conservation scores (top left) for each a.a. or cross-section of surface rendering of BLTP3A channel (top right) highlighting hydrophobic residues (orange). Linear representation of BLTP3A: per residue ConSurf scores (top) and RBG organization (bottom). IDR predicted intrinsically disordered region (light gray), CH C-terminal helix (dark gray). ( B ) Alignment of the a.a. of motifs important for the indicated protein interactions of BLTP3B with corresponding sequences of BLTP3A. ConSurf conservation scores for each a.a. is indicated by color (same color scheme as in ( A )). ( C ) Western blot of lysates of wild-type mouse tissues for BLTP3A and vinculin as a loading control. ( D ) Western blot of control and edited (BLTP3A^V5) cell clones for BLTP3A, V5, and alpha-tubulin as a loading control. ( E ) Fluorescence images of endogenously edited (left) or parental control (middle) A549 cells with antibodies against LAMP1 (green) and V5 (magenta). Scale bar, 10 μm. The insets are a zoom of a small region of the cell. Scale bar, 1 μm. Right: Fluorescence of exogenous BLTP3A-GFP (green) in endogenously edited A549 cell. Scale bar, 5 μm. Insets: zoom of square region of cell showing co-localization of endogenous BLTP3A^V5 signal from immunolabeling with antibodies against V5 (magenta) and BLTP3A-GFP fluorescence (green). Scale bar, 1 μm. ( F ) Fluorescence image of wild-type RPE-1 cell immunostained with antibodies against LAMP1 (green) and DAPI (blue). Scale bar, 5 μm. ( G ) Fluorescence image of an RPE-1 cell expressing exogenous BLTP3A-mRFP (large field, inverted grays) and LAMP1-GFP (not shown). Scale bar, 5 μm. The area enclosed by a dotted rectangle is shown at right at high magnification with BLTP3A-mRFP in magenta and LAMP1-GFP in green (individual channels are shown as inverted grays). Red arrows indicate large BLTP3A accumulations not associated with lysosomes. Scale bar, 1 μm. ( H ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles, many of them tethered to the surface of lysosomes. Scale bar, 500 nm. ( I ) Distance between the membranes of lysosomes and tethered vesicles from EM micrographs. Mean = 10.8 nm; standard error of the mean = ±0.20 nm. ( J ) Left: Fluorescence image of an RPE-1 cell expressing exogenous BLTP3A-mRFP (inverted grays) and immunolabeled with antibodies against VAMP7 (shown in the high mag fields at right). Scale bar, 5 μm. Right: zooms of different RPE-1 cells expressing exogenous BLTP3A-mRFP (magenta) and immunolabeled (green) with antibodies against the following endogenous proteins, VAMP4 and ATG9A. Individual channels are shown as inverted grays. Merge of channels on bottom. Scale bar, 1 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Residue, Western Blot, Control, Clone Assay, Fluorescence, Immunolabeling, Expressing

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Live fluorescence images (inverted grays) of RPE-1 cells expressing either GFP-Rab45 (left), BLTP3A-mRFP (center), or both proteins together (only BLTP3A is shown) (right) as indicated. Scale bar, 5 μm. High-magnification scale bar, 2 μm. ( B ) Genomic sequence of the edited BLTP3A locus (insertion of the V5 epitope) in A549 cell. Blue, small Gly-Ser linkers; green, V5 epitope sequence. ( C ) AlphaFold prediction of BLTP3A. The site where the V5 epitope (V904) was inserted is indicated. The long disordered sequence and the C-terminal helix are shown in gray. ( D ) Left: Fluorescence image of an RPE-1 cell expressing exogenous BLTP3B-mRFP (inverted grays) and immunolabeled with antibodies against endogenous VAMP7 (shown at right in the high magnification of the squared region in the main field). Scale bar, 5 μm. Right: zooms of different RPE-1 cells expressing exogenous BLTP3B-mRFP (magenta) and immunolabeled with antibodies (green) against endogenous VAMP4 or ATG9A. Individual channels are shown as inverted grays. Merge of channels on bottom. Scale bar, 1 μm. ( E ) Fluorescence images of RPE-1 cells expressing the indicated BLTP3A-mRFP construct. Scale bar, 5 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Expressing, Sequencing, Immunolabeling, Construct

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images of RPE-1 cells expressing exogenous BLTP3A-mRFP (shown in inverted grays) and (not shown) GFP-tagged wild-type Rab7 (left) or dominant negative (DN) Rab7 (T22N) (right). Scale bar, 5 μm. ( B ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell expressing GFP-tagged dominant negative Rab7. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles. Scale bar, 500 nm. ( C ) BLTP3 chimeras design. Left: Surface representation of the predicted RBG core of BLTP3A. Red and blue indicate positive and negative charges, respectively, and gray indicates hydrophobic surfaces. Right: Surface representation (top) and ribbon representation (bottom) of the “untwisted” protein showing individual RBG motifs. Bottom: Cartoon of chimeras consisting of BLTP3A (dark orange) and BLTP3B (light orange) RBG motifs. ( D ) High-magnification live fluorescence images of RPE-1 cells expressing the indicated BLTP3-mRFP constructs (magenta) and LAMP1-GFP (green). Individual channels are shown as inverted grays. Scale bar, 1 μm. ( E ) Ribbon representation of the AlphaFold prediction of a.a. 1–336 of BLTP3A. Blue indicates loops connecting adjoining RBG motifs, and gray indicates the first beta-strand of the third RBG motif. ( F ) Fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-1-336-mRFP and either (not shown) GFP-Rab7 (left), or GFP-Rab7 T22N (right). Scale bar, 5 μm. A zoom of an area of the cell at left (dotted square) expressing BLTP3A-1-336-mRFP (magenta) is also shown, along with the Rab7 fluorescence (green), demonstrating the localization of BLTP3A-1-336-mRFP around the entire profile of lysosomes. Individual channels are shown as inverted grays. Scale bar, 2 μm. ( G ) Cartoon depicting the proposed association of BLTP3A vesicle clusters with the surface of lysosomes and the dependence of this association on Rab7.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Expressing, Dominant Negative Mutation, Construct

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Top: Linear representation of BLTP3A RBG organization and C-terminal truncations indicated by arrows. Bottom left: AlphaFold-based structure of the C terminus of BLTP3A channel. Individual residues are colored by conservation scores as Fig. . Truncations are indicated by arrows. Bottom right: Fluorescence images (inverted grays) of RPE-1 cells expressing the indicated BLTP3A-mRFP constructs along with dominant negative Rab7 (not shown) demonstrating that the property of BLTP3A to bind and cluster vesicles is dependent on its region comprised between a.a. 1327 and 1364. Note that the construct 1–1327, shows a focal accumulation next to the nucleus, which is VAMP7 negative, likely reflecting its pool bound to Rab45 (see Fig. ). Scale bar, 5 μm. Zoomed images (dotted squares) are shown below the main field along with VAMP7 fluorescence. Scale bar, 1 μm. ( B ) Western blots of cell extracts (inputs) of control and edited A549 cells, and of material immunoisolated from these extracts by anti-V5 magnetic beads. Immunolabeling for BLTP3A, V5 (endogenously tagged BLTP3A), and for GAPDH as a loading control, are shown. ( C ) Scatter plot of mass spectrometry-identified proteins in immunoisolated material from either control or endogenously edited BLTP3A^V5 A549 cells using anti-V5 magnetic beads ( N = 3, biological replicates). Proteins significantly enriched in material immunoisolated form edited cells compared to wild-type cells are plotted in the right-top quadrant. Proteins of note are labeled in magenta.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Expressing, Construct, Dominant Negative Mutation, Western Blot, Control, Magnetic Beads, Immunolabeling, Mass Spectrometry, Labeling

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3A-GFP and mApple-IST1 before and after addition of LLOMe. Arrowheads (magenta) point to BLTP3A accumulations shed from lysosomes upon addition of LLOMe. Scale bar, 5 μm. ( B ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker NPC1-GFP (green). Fluorescence of individual channels is shown in inverted grays. Scale bar, 2 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Marker

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker LAMP1-GFP (green) expressed in wild-type A549 cells before and after addition of LLOMe. Fluorescence of individual channels is shown in inverted grays. Scale bar, 5 μm. ( B ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker LAMP1-GFP (green) expressed in VPS13C KO A549 cells before and after addition of LLOMe. Fluorescence of individual channels is shown in inverted grays. Scale bar, 5 μm. ( C ) Live fluorescence images (inverted grays) of RPE-1 cells expressing exogenous GFP-LRRK1 K746G (left) and BLTP3A-mRFP (right). A partial association of BLTP3A-mRFP and GFP-LRRK1 K746G was observed. Scale bar, 10 μm. ( D ) Western blot of lysate of RPE-1 cells expressing exogenous RFP-LRRK1 K746G or RFP-LRRK1 D1409A for RFP (to detect LRRK1 fusions), Rab7, phospho-Rab7 S72, and alpha-tubulin as a loading control. Individual lanes are biological replicates.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Marker, Expressing, Western Blot, Control

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Alignment of the region of BLTP3A orthologues from different species centered on the a.a. region required for mATG8 binding in human BLTP3A. The alignment shows a high degree of conservation of the key residues of the LC3-interacting region (LIR) motif among several chordates and also observed in flies. ( B ) Live fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-mRFP (top) or GFP-LC3B (bottom) in either fed (left) or starved (right) conditions. Arrows (magenta) indicate GFP-LC3B positive foci. Scale bar, 5 μm. ( C ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3A-mRFP and GFP-LC3B before and after addition of LLOMe. Arrowheads point to lysosomes where BLTP3A and LC3B decorate the entire lysosome profile upon addition of LLOMe. Scale bar, 5 μm. ( D ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3AΔLIR-mRFP and GFP-LC3B before and after addition of LLOMe. Arrowheads point to lysosomes where LC3B, but not BLTP3A lacking a LIR motif, decorates the entire lysosome profile upon addition of LLOMe. Scale bar, 5 μm. ( E ) Correlative fluorescence-FIB-SEM microscopy of GFP-LC3B and BLTP3A-mRFP-positive lysosomes in an RPE-1 cell 15 min after LLOMe addition. Left: Fluorescence image of an RPE-1 cell expressing GFP-LC3B (green), BLTP3A-mRFP (magenta), and mito-BFP (not shown), 15 min after addition of LLOMe. Scale bar, 5 μm. Region of cell for FIB-SEM marked by dotted line. Middle: High magnification of the RPE-1 cell at left (blue square) showing both the BLTP3A-mRFP (magenta) and the GFP-LC3B (green) channels. Individual channels are shown as inverted grays. Merged channel also shows mito-BFP (blue) used for aligning EM and fluorescence images. Scale bar, 2 μm. Right: EM reconstruction (scale bar, 1 μm) corresponding to the blue square from left. Lysosome, dark green; ER, yellow; mitochondria, blue; small vesicles, magenta.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Binding Assay, Fluorescence, Expressing, Microscopy

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) AlphaFold3 multimer prediction of full-length MAP1LC3B (green) and a.a. 1110–1150 of BLTP3A (magenta). Arrows indicate key residues of the LIR motif of BLTP3A. ( B ) AlphaFold3 multimer predictions of mATG8 proteins and a.a. 1110–1150 of BLTP3A with and without the LIR motif (ΔLIR). ( C ) Time-series of live fluorescence images (inverted grays) of BLTP3A-mRFP and GFP-LC3B before and after addition of GPN. Arrowheads point to lysosomes where BLTP3A and LC3B decorate the entire profile upon addition of GPN. Time, seconds. Scale bar, 5 μm. ( D ) Genomic sequence of the edited BLTP3A locus in A549 cell. Blue, gRNA; green, PAM; red, indel mutations. ( E ) Quantification of relative LAMP1 expression from western blots ( N = 3, biological replicates) of Fig. . Error bars indicate the standard error of the mean (SEM).

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Sequencing, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Brightfield image of RPE-1 cells incubated with MSU crystals (200 μg/mL) for 2 h. Arrowheads indicate MSU crystals. Scale bars, 5 μm. ( B ) Live fluorescence images of RPE-1 cells expressing BLTP3A-mRFP and LAMP1-GFP (shown as inverted greys) and incubated with MSU crystals for 2 h. Scale bar, 5 μm. Zoomed images (dotted rectangles) of individual channels are shown to the right. Scale bars, 2 μm. Arrowheads point to the surface of crystal-containing vacuoles positive for BLTP3A-mRFP and LAMP1-GFP. ( C ) Live fluorescence images of RPE-1 cells co-expressing either BLTP3A-mRFP (top row) or BLTP3AΔLIR-mRFP (bottom row) with GFP-LC3B and incubated with MSU crystals for 2 h. Individual channels are shown as inverted greys. Scale bar, 10 μm. Zoomed images (dotted rectangles) of individual channels are shown to the right. Scale bar, 2 μm. Arrowheads point to the surface of the crystal-containing vacuoles positive for GFP-LC3B to highlight the presence of BLTP3A-mRFP (top) and the absence of BLTP3AΔLIR-mRFP (bottom). ( D ) Time-series of live fluorescence images (inverted grays) of BLTP3A-mRFP and GFP-LC3B in RPE-1 cells incubated with MSU crystals. Scale bar, 2 μm. Arrowheads point to the surface of crystal-containing vacuoles.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Incubation, Fluorescence, Expressing

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Western blots of cell extracts (inputs) of control and edited A549 cells (BLTP3A KO clones) for the proteins indicated. ( B ) Fluorescence images of parental control (left) or BLTP3A KO (right) A549 cells with antibodies against LAMP1 (green). Scale bar, 20 μm. ( C ) Fluorescence images of parental control (top row) or BLTP3A KO (bottom row) A549 cells with antibodies against galectin-3 (inverted greys). Cells were treated with vehicle control (left column) or 1 mM LLOMe (right column). Scale bar, 10 μm. ( D ) Quantification of galectin-3 spots per cell from field ( C ) ( N = 3, biological replicates). Error bars report the standard error of the mean (SEM). ** P < 0.01. Mean number of galectin-3 spots per cell and number of cells counted per condition indicated.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Western Blot, Control, Clone Assay, Fluorescence

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images (inverted greys) of BLTP3A KO A549 cells expressing indicated RFP protein (top row) with antibodies against galectin-3 (bottom row). Dotted magenta line indicates cell boundary. Cells were treated with vehicle control (left column) or 1 mM LLOMe (right three columns). Scale bar, 10 μm. ( B ) Quantification of galectin-3 spots per cell from field ( A ) ( N = 3, biological replicates.). Error bars report the standard error of the mean (SEM). *** P < 0.001; ** P < 0.01; * P < 0.05; n.s., not significant. Mean number of galectin-3 spots per cell and number of cells counted per condition indicated.

Article Snippet: pmCh-N1 BLTP3A 1-1327 , De Camilli lab; this manuscript , RRID:Addgene_241253.

Techniques: Fluorescence, Expressing, Control

Journal: Nature communications

Article Title: Molecular insights into degron recognition by CRL5 ASB7 ubiquitin ligase.

doi: 10.1038/s41467-024-50556-x

Figure Lengend Snippet: Fig. 4 | ITC analysis of the key elements required for ASB7 recognition. a ITC fitting curves of LZTS1 peptide titrated to wild-type ASB7 and site 1 mutants. NB, no apparent binding under our experimental conditions. b ITC fitting curves of LZTS1 peptide titrated to wild-type ASB7 and site 2/3 mutants. c ITC fitting curves of LZTS1

Article Snippet: Protein expression and purification The human ASB7 (residues 11–318) was amplified via PCR from a human cDNA library and seamlessly cloned into the pET28MKH8SUMO vector (Addgene plasmid #79526).

Techniques: Binding Assay

Journal: Aging (Albany NY)

Article Title: MicroRNA 27b promotes cardiac fibrosis by targeting the FBW7/Snail pathway

doi: 10.18632/aging.102465

Figure Lengend Snippet: miR-27b modulated the expression of Snail by inhibiting its degradation ( A , B ) The mRNA level of Snail in CFs treated with AngII combined with miR-27i ( A ) or miR2-7b ( B ). ( C ) The effect of miR-27b on the expression of Snail in CFs treated with cycloheximide (CHX, 1 μg/ml) at indicated time points. ( D ) Roles of miR-27b in Snail ubiquitination. ( E ) Characterization of the messenger RNA (mRNA) of FBW7, which depicted miR-27b binding site (BS) in its 3′-untranslated region (UTR). ( F ) The interaction of FBW7 and Snail in CFs. ( G ) The effect of FBW7 overexpression on the expression of Snail in CFs treated with cycloheximide (CHX, 1 μg/ml) at indicated time points. ( H ) The effect of FBW7 overexpression on the ubiquitination of Snail. Arrow indicates the GFP-FBW7. Data were represented as mean ± SEM (n=4). N, p >0.05.

Article Snippet: The 3′UTR of the FBW7 luciferase reporter plasmid (pRL-TK 3′FBXW7 UTR) were obtained from Addgene (#26649).

Techniques: Expressing, Ubiquitin Proteomics, Binding Assay, Over Expression

Journal: Aging (Albany NY)

Article Title: MicroRNA 27b promotes cardiac fibrosis by targeting the FBW7/Snail pathway

doi: 10.18632/aging.102465

Figure Lengend Snippet: FBW7 was the miR-27b target in cardiac fibrosis ( A – C ) The mRNA levels of FBW7 in serum-treated CFs ( A ), AngII ( B ), or miR-27b ( C ). ( D ) Snail expression in CFs transfection with miR-27b and/or peGFP-FBW7 plasmids. ( E , F ) The proliferation of CFs treated in ( D ) were analyzed by MTT test ( E ), and BrdU assay ( F ). ( G ) CFs transfected with miR-27i and/or FBW7 siRNA were subjected to AngII treatment as indicated. Snail expression was analyzed using WB. ( H , I ) The proliferation of CFs treated in ( G ) was analyzed by MTT test ( H ), and BrdU assay ( I ). Data were represented as mean ± SEM (n=4). *, p <0.05; **, p <0.01.

Article Snippet: The 3′UTR of the FBW7 luciferase reporter plasmid (pRL-TK 3′FBXW7 UTR) were obtained from Addgene (#26649).

Techniques: Expressing, Transfection, BrdU Staining

Journal: Aging (Albany NY)

Article Title: MicroRNA 27b promotes cardiac fibrosis by targeting the FBW7/Snail pathway

doi: 10.18632/aging.102465

Figure Lengend Snippet: Antagomir-27b attenuated cardiac fibrosis in rat model of MI ( A ) Real-time PCR results of miR-27b levels in miR-27i or saline-treated specimens (3 weeks post-injection). ( B , C ) Analytical results of miR-27i-treated peri-infarct area of rat heart (3 weeks post-treatment). ( B ) Typical heart sections after treatments of Masson trichrome staining, laminin and collagen I immunostaining. Scale bar, 20 μm. ( C ) The percentage of tissue area represented the deposition of collagen I, where the automated image analyzer was used for its quantification. ( D ) Quantitative reverse transcription–PCR results of collagen I, collagen III, and MMP-9 mRNA levels. ( E ) FBW7 and Snail expression in rat heart. Data were represented as mean ± SEM (n=6). *, p <0.05; **, p <0.01.

Article Snippet: The 3′UTR of the FBW7 luciferase reporter plasmid (pRL-TK 3′FBXW7 UTR) were obtained from Addgene (#26649).

Techniques: Real-time Polymerase Chain Reaction, Saline, Injection, Staining, Immunostaining, Reverse Transcription, Expressing

Journal: Nature Communications

Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

doi: 10.1038/s41467-021-22677-0

Figure Lengend Snippet: a Preassembled GST-IQGAP1-DBR and His 6 -DIAPH1-NT complexes were incubated with MBP-CLIP170-NT. In parallel preassembled GST-IQGAP1-DBR and MBP-CLIP170-NT complexes were incubated with His 6 -DIAPH1-NT. After reisolating GST-IQGAP1-DBR, the amount of co-purifying MBP-CLIP170-NT or His 6 -DIAPH1-NT was determined. NT = N-terminus, DBR = Diaphanous Binding Region. b Schematic representing inferred IQGAP1 interaction dynamics.

Article Snippet: The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table .

Techniques: Incubation, Binding Assay

Journal: Nature Communications

Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

doi: 10.1038/s41467-021-22677-0

Figure Lengend Snippet: a Time-lapse images of GFP-CLIP170 during cell division. b Proximity ligation assays (PLA) mapping the proximity DIAPH1 to IQGAP1 (top), IQGAP1 to CLIP170 (middle) and CLIP170 to DIAPH1 (bottom) during mitotic progression. c Quantitation of PLA foci in metaphase and anaphase * p < 0.0001 using a nonparametric two-tailed Mann-Whitney t -tests for three experimental repeats, n = 50 cells examined over three independent experiments. d Proximity ligation assays (PLA) mapping the proximity DIAPH1 to IQGAP1 and IQGAP1 to CLIP170 in Centrinone B-treated cells. e Quantitation of PLA foci in d . At cell poles lacking astral microtubules, DIAPH1 and IQGAP1 are in close proximity but IQGAP1 and CLIP170 are not. * p = 0.0006, ** p < 0.0001 using nonparametric two-tailed Mann-Whitney t -tests for three experimen t al repeats, n = 50 cells examined over three independent experiments. Scale bars are 10 μm.

Article Snippet: The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table .

Techniques: Ligation, Quantitation Assay, Two Tailed Test, MANN-WHITNEY