Journal: Science Advances

Article Title: RAB5c orchestrates LC3-associated phagocytosis to promote microbicidal function of macrophages

doi: 10.1126/sciadv.adz0196

Figure Lengend Snippet: ( A ) mCh-RAB5c MFI at phagosomes ( n = 12) of RAW264.7 cells fed Op-zym (see movie S1). ( B ) CLEM analysis of Op-zym + phagosome in RAW264.7-mCh-RAB5c cells. Left: Fluorescence image. Middle: overlay (bottom) and corresponding TEM cross section (top). Right: Magnified inset; arrows indicate single membrane. Scale bars, 5 μm (left and middle) and 1 μm (right). ( C ) Confocal images of RAW264.7-mCh-RAB5c cells stimulated with Op-zym, IgG-beads, or BSA-beads for 5 min. Insets fluorescence intensity (show as RAINBOW LUT). Scale bar, 5 μm. DIC, differential interference contrast. ( D and E ) Confocal images (D) and mCh-LC3B MFI at phagosomes (E) for RAW246.7 cells expressing scrambled shRNA (sh_Ctrl) or indicated shRNAs (#1; #2) fed Op-zym. Scale bar, 5 μm. ( F to H ) Confocal images (F) and MFI of LC3 at phagosomes for RAW264.7 cells (G) and BMDMs (H) expressing sh_Ctrl or sh_ Rab5c fed Op-zym. Scale bars, 5 μm. ( I ) Immunoblot of LC3A/B, RAB5a/b/c, EEA1, UNC93B1 (loading control), and GM130 (purification control) in IgG-bead– or BSA-bead–containing phagosomes purified from RAW264.7 cells expressing sh_Ctrl or sh_ Rab5c . M r , relative molecular mass. ( J and K ) Confocal images (shown as FIRE LUT) (J) and LAMP1 MFI at phagosomes (K) for RAW264.7 cells fed Op-zym. Scale bar, 10 μm. ( L and M ) MFI (L) and percentage (M) of Op-pHrodo-zym in RAW264.7 cells analyzed by flow cytometry. ( N ) MFI of opsonized DQ-BSA–coated beads in RAW264.7 cells. Colored symbols [(A), (E), (G), (H), and (K)] or bars [(L) to (N)] represent means of biological replicates, each indicated as a gray (phagosome) or white (sample) object. Shaded area (A) or error bars, ±SEM. Statistical comparison between groups are unpaired Student’s t test. Data are pooled from two (A) or three [(E), (G), (H), and (K)] independent experiments or are representative of four (I), three [(L) and (M)], or two (N) independent experiments. *, phagosomes; N, nuclei.

Article Snippet: RAW264.7 macrophage cell line (ATCC, SC-6003), HEK PEAKrapid cells (ATCC, CRL-2828), and mouse NIH 3T3 fibroblasts expressing human colony-stimulating factor 1 (3T3-MCSF) cells were provided by D. Zamboni (Faculdade de Medicina de Ribeirão Preto).

Techniques: Fluorescence, Membrane, Expressing, shRNA, Western Blot, Control, Purification, Flow Cytometry, Comparison

Journal: Science Advances

Article Title: RAB5c orchestrates LC3-associated phagocytosis to promote microbicidal function of macrophages

doi: 10.1126/sciadv.adz0196

Figure Lengend Snippet: RAW264.7 cells or BMDMs expressing sh_Ctrl or sh_ Rab5c . ( A ) Immunoblot of class III PI(3)K complex in BSA-bead– or IgG-bead–containing phagosomes from RAW264.7 cells. ( B and C ) Confocal images (shown as SMART LUT) (B) and p40 phox -PX-Venus MFI at phagosomes ( n = 18) (C) in RAW264.7-p40 phox -PX-Venus cells fed Op-zym. *, phagosomes (see movie S3). ( D and E ) Immunoblot (D) and quantification (E) of LC3A/B-II in RAW246.7 cells ± Op-zym (25 min), C8 (2 μM; 25 min), DMXAA (50 μg/ml; 1 hour), and PP242 (1 μM; 2 hours). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), loading control. NS, nonstimulated. ( F ) Immunoblot of NOX2 components in IgG-bead– or BSA-bead–containing phagosomes from RAW264.7 cells. ( G to I ) NBT assay in macrophages fed Op-zym. DIC images (G) and percentage of NBT + phagosomes in RAW264.7 cells (H) and BMDMs (I). Arrowheads, NBT + (red) or NBT − (yellow). ( J to L ) Confocal images (J) and p-p47 phox MFI at phagosomes in RAW264.7 cells fed Op-zym for 15 (K) or 40 (L) min. ( M and N ) Confocal images (M) and MFI (N) of p-ERK1/2 in RAW264.7 cells fed Op-zym (15 min). ( O ) Percentage of NBT + phagosomes in RAW264.7 cells ± ERK1/2 inhibitor (100 μM) for 10 min before Op-zym. Colored symbols [(C), (K), (L), and (N)] or bars [(E), (H), (I), and (O)] represent means of biological replicates, each indicated as a gray (phagosome) or white (sample) object. Shaded area (C) or error bars, ±SEM. Statistical comparisons are unpaired Student’s t test [(C), (E), (H), (I), (K), (L), and (N)] or analysis of variance (ANOVA) and Tukey’s multiple comparisons (O). Data are pooled from two (C) or three [(E), (K), (L), and (N)] independent experiments or are representative of three to five [(A) and (F)], two [(B), (I), and (O)], or three [(D), (G), and (H)] independent experiments. Scale bars, 10 μm.

Article Snippet: RAW264.7 macrophage cell line (ATCC, SC-6003), HEK PEAKrapid cells (ATCC, CRL-2828), and mouse NIH 3T3 fibroblasts expressing human colony-stimulating factor 1 (3T3-MCSF) cells were provided by D. Zamboni (Faculdade de Medicina de Ribeirão Preto).

Techniques: Expressing, Western Blot, Control

Journal: Science Advances

Article Title: RAB5c orchestrates LC3-associated phagocytosis to promote microbicidal function of macrophages

doi: 10.1126/sciadv.adz0196

Figure Lengend Snippet: RAW264.7 cells expressing sh_Ctrl or sh_ Rab5c . ( A and B ) Confocal images (A) and MFI (B) of ATP6V1A at phagosomes in cells fed Op-zym. ( C ) Immunoblot of components of the V-ATPase complex and ATG8 lipidation machinery in purified BSA-bead– or IgG-bead–containing phagosomes. ( D ) ATP6V0a3-EGFP MFI at phagosomes ( n = 20) of cells fed Op-zym (see movie S4). ( E ) Enhanced-resolution confocal image of cells expressing ATP6V0a3-EGFP and mCh-RAB5c. Left: Maximal-intensity projection. Middle: Single Z -sections of insets a and b. Right: Three-dimensional projections of insets a and b. ( F ) Immunoblot of endosome immunoprecipitation (EndoIP) from RAW264.7-3X-FLAG-RAB5c cells. Loaded fractions correspond to 1.3% of input and flow-through (FT) and 20% of immunoprecipitation (IP) eluate. ( G ) Immunoblot of coimmunoprecipitation (co-IP) from RAW264.7 cells overexpressing 3X-FLAG-mCherry or 3X-FLAG-RAB5c. Loaded fractions correspond to 2% of input and FT and 20% of IP eluate. Blots were probed with the indicated antibodies. ( H ) LC3 MFI at phagosomes upon treatment with DPI (0.61 or 5 μM) before Op-zym. ( I ) LC3 MFI at phagosomes upon treatment with DPI (5 μM) or DPI and chloroquine (CQ; 100 μM) before Op-zym. Colored symbols [(B), (D), (H), and (I)] represent means of biological replicates (phagosome), each indicated as a gray object. Shaded area (D) or error bars, ±SEM. Statistical comparison between groups are unpaired Student’s t test [(B) and (D)] or ANOVA and Tukey’s multiple comparisons [(H) and (I)]. Data are pooled from three [(B), (H), and (I)] or two (D) independent experiments or are representative of three [(A) and (F)], four to five (C), or two [(E) and (G)] independent experiments. Scale bars, 5 μm.

Article Snippet: RAW264.7 macrophage cell line (ATCC, SC-6003), HEK PEAKrapid cells (ATCC, CRL-2828), and mouse NIH 3T3 fibroblasts expressing human colony-stimulating factor 1 (3T3-MCSF) cells were provided by D. Zamboni (Faculdade de Medicina de Ribeirão Preto).

Techniques: Expressing, Western Blot, Purification, Immunoprecipitation, Co-Immunoprecipitation Assay, Comparison

Journal: bioRxiv

Article Title: Analysis of motor-based transport in primary cilia by dynamic mode decomposition of live-cell imaging data

doi: 10.64898/2026.03.27.714708

Figure Lengend Snippet: (A, B) 2D (A) and 3D (B) SIM imaging of Halo-KIF13B (red) and IFT172-eGFP (green) stably expressed in hTERT-RPE1 cells subjected to 24 hours of starvation. In panel B, the white line was used to calculate fluorescence intensity profiles. Scale bars are 2 µm. (C) Fluorescence intensity profile calculated for the same cilium as in (B), drawing a line near the ciliary base, perpendicular to the axoneme. (D) Confocal (right) and 2D STED (left) images of fixed hTERT-RPE1 cells expressing Halo-KIF13B (red) and IFT172-eGFP (gray) after 24 hours of starvation, with FBF-1 antibody staining (cyan). Here, a Gaussian blur with a sigma of 2 pixels is used to smooth the images. Scale bars are 1 µm.

Article Snippet: The hTERT-RPE1 parental cell line stably expressing IFT172-eGFP (derived from the immortalized hTERT-RPE1 cell line, ATCC, clone CRL-4000) has been described previously ( ; ). hTERT-RPE1 stably co-expressing IFT172-eGFP and Halo-KIF13B were generated by transducing the aforementioned cell line with lentiviral particles expressing pCDH-EF1aGW-IRES-Blast-Halo-KIF13B plasmid.

Techniques: Imaging, Stable Transfection, Fluorescence, Expressing, Staining

Journal: bioRxiv

Article Title: O-GlcNAcylation regulates PPAR-driven metabolic programming in intestinal stem cells

doi: 10.64898/2026.03.13.711696

Figure Lengend Snippet: (A) Quantification and representative flow plots of Lgr5 -eGFP hi ISCs of vehicle and ST-treated organoids derived from ( Ppar-d/a iKO : Ppard fl/fl ; Ppara fl/fl ; Vil CreER ; Lgr5 eGFP-IRES-CreER ) and wild type controls (WT: Vil CreER ; Lgr5 eGFP-IRES-CreER ). N=4, ns: not significant, *p<0 . 05 by two-way ANOVA. (B) Quantification of BRDU + GFP hi ISCs per organoid in vehicle and ST treated Ppar-d/a iKO intestinal organoids and representative immunofluorescent images Magenta: BRDU. Green: GFP, White: DAPI. N=3. ns: not significant by Students t-test (C) Immunoblot of PPAR target proteins in WT and Ppar-d/a iKO intestinal organoids treated with ST. (D) Quantification and representative images of organoids formed per sorted Lgr5 -eGFP hi ISC from WT and Ppar-d/a iKO animals treated with either vehicle or ST. N=5. ns: not significant, *p<0 . 05 by two-way ANOVA. (E) Immunoblot analysis of input and anti-HA, anti-IgG and anti-OGT immunoprecipitate from WT and HA-PPARd expressing MC38 cells. (F) Immunoblot analysis of input and anti-HA immunoprecipitate of HA-PPARd MC38 cells treated with vehicle or ST.

Article Snippet: MC38- Ppard- KO and parental wild-type MC38 cell lines were obtained from Ubigene.

Techniques: Derivative Assay, Western Blot, Expressing