Journal: bioRxiv

Article Title: Analysis of dRIF Composition Identifies ZNF346 as a Regulator of PKR Activation

doi: 10.64898/2026.06.03.729416

Figure Lengend Snippet: ( A-F ) Representative fluorescence microscopy images for in situ validation of dRIF candidate proteins identified by MS. Validation of candidates was performed by either immunostaining for the target protein or by expression of eGFP-tagged derivative of the target protein in A549 mApple-PKR K296R cells, as indicated in the figure labels. Percentages indicate the frequency with which dRIF candidate proteins colocalized with mApple-PKR K296R foci by fluorescence microscopy. For validation experiments, cells were transfected with in vitro -transcribed dsRNA to confirm that recruitment of candidate proteins to dRIFs is stimulated by dsRNA and is not specific to poly(I:C).

Article Snippet: Cell lines expressing EF1-eGFP-ZNF346 and Tet-On-3G-eGFP-ZNF346 were generated from an A549 WT parental cell line purchased from ATCC (CCL185).

Techniques: Fluorescence, Microscopy, In Situ, Biomarker Discovery, Immunostaining, Expressing, Transfection, In Vitro

Journal: bioRxiv

Article Title: Analysis of dRIF Composition Identifies ZNF346 as a Regulator of PKR Activation

doi: 10.64898/2026.06.03.729416

Figure Lengend Snippet: ( A ) Schematic of siRNA knockdown strategy to test the impact of depleting dRIF resident proteins on dRIF formation. A549 PKR KO mApple-PKR K296R cells were treated with siRNA for 48 hours prior to dsRNA transfection. siRNA-treated cells were transfected with dsRNA for 4 hours and fixed for analysis by fluorescence microscopy. siRNA-treated cells were harvested in parallel for western blotting to assess knockdown efficiency. ( B ) Representative fluorescence microscopy images of cells with dRIFs (proxied by mApple-PKR K296R foci) in each siRNA knockdown condition. ( C ) Quantification showing the number of dRIFs per cell, denoted by mApple-PKR K296R -positive foci, in each siRNA knockdown condition. The mean values for each biological replicate (calculated as the mean of per-image means for each rep) are shown as circles for each condition. Statistical comparisons were made using an unpaired two-tailed t-test on the per-image means for each siRNA knockdown condition across biological replicates. Data was obtained from 3-5 independent experiments per condition, 3+ images per experiment, and 50+ cells per biological replicate per condition. ( D ) Quantification of cy5-labeled dsRNA fluorescence intensity (normalized to mApple-PKR K296R fluorescence intensity) in cells treated with siRNA targeting DHX9 (siDHX9) or a non-targeting control (siScramble). The per-image means are shown as larger circles, smaller circles represent individual data points. Statistical comparisons were made using a Wilcoxon rank-sum test on per-image means. Data was obtained from 2 independent experiments and 40+ cells and 3+ fields-of-view per condition and biological replicate. ( E ) Microscopy images measuring dsRNA fluorescence intensity (normalized to mApple-PKR K296R fluorescence intensity) in cells treated with siRNA targeting DHX9 (siDHX9) or a non-targeting control (siScramble). ( F ) Fluorescence intensity profiles from the lines in 5E) showing the relative enrichment of cy5-dsRNA and mApple-PKR K296R in dRIFs in single cells treated with siRNA targeting DHX9 (siDHX9) or a non-targeting control (siScramble). All data in this figure shows cells transfected with 400 ng/mL dsRNA. All statistical analyses in this figure use the following p-value cutoffs: **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, n.s. P > 0.05.

Article Snippet: Cell lines expressing EF1-eGFP-ZNF346 and Tet-On-3G-eGFP-ZNF346 were generated from an A549 WT parental cell line purchased from ATCC (CCL185).

Techniques: Knockdown, Transfection, Fluorescence, Microscopy, Western Blot, Two Tailed Test, Labeling, Control

Journal: bioRxiv

Article Title: Analysis of dRIF Composition Identifies ZNF346 as a Regulator of PKR Activation

doi: 10.64898/2026.06.03.729416

Figure Lengend Snippet: ( A ) Representative fluorescence microscopy images showing the relationship between PKR activation (visualized with an -p-PKR antibody), dRIF formation (denoted by mApple-PKR foci) and the presence of stress granules (denoted by GFP-G3BP1 foci) in dsRNA-transfected RNase L KO cells treated with siRNA targeting ZNF346 (siZNF346) or a non-targeting control (siScramble). ( B ) Quantification showing the mean intensity of p-PKR staining (normalized to mApple-PKR signal) in dsRNA-transfected RNase L KO cells treated with siRNA targeting ZNF346 (siZNF346) or a non-targeting control (siScramble). Statistical comparisons were made by performing an unpaired two-tailed t-test on per-image means. Data was obtained from 5 independent experiments and 300+ cells and 2+ fields-of-view per biological replicate and condition. ( C ) Quantification showing the percentage of stress granule-positive cells (proxied by GFP-G3BP1-positive foci) in dsRNA-transfected RNase L KO cells treated with siRNA targeting ZNF346 (siZNF346) or a non-targeting control (siScramble). Statistical comparisons were made by performing an unpaired two-tailed t-test on per-image means. Data was obtained from 5 independent experiments and 300+ cells and 5+ fields-of-view per biological replicate and condition. ( D ) Schematic for Tet-ON expression system for inducible eGFP-ZNF346 expression in A549 WT cells. ( E ) Temporal schematic for doxycycline-induced expression of eGFP-ZNF346 in A549 WT cells. ( F ) Representative fluorescence microscopy images showing doxycycline-induced expression of eGFP-ZNF346 and immunofluorescence staining for p-PKR in A549 WT cells treated with 2 ug/mL doxycycline with or without 400 ng/mL dsRNA treatment. ( G ) Quantification of the mean p-PKR immunofluorescence intensity in cells expressing eGFP-ZNF346 and treated with 2 ug/mL doxycycline with or without 400 ng/mL dsRNA treatment. Cells were binned according to eGFP nuclear fluorescence intensities, where eGFP-high cells are the top 50% and eGFP-low cells are the bottom 50% of single cells expressing eGFP-ZNF346. Circles represent individual data points. Data was obtained from 50+ cells for the no-dsRNA condition and 90+ cells for the dsRNA-treated condition. ( H ) Representative fluorescence microscopy images comparing the effects of expressing either eGFP tagged with V5 or eGFP-ZNF346 on translation arrest (proxied by α -puromycin staining intensity) in A549 WT cells transfected with 400 ng/mL dsRNA. ( I ) Quantification of mean -puromycin intensity in A549 WT cells expressing either eGFP tagged with V5 or eGFP-ZNF346 and transfected with 400 ng/mL dsRNA. Statistical comparisons were made using a Wilcoxon rank-sum test on individual data points. Data was obtained from 2 independent experiments and 150+ cells per biological replicate. All data in this figure shows cells transfected with 400 ng/mL dsRNA. All statistical analyses in this figure use the following p-value cutoffs: **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, n.s. P > 0.05.

Article Snippet: Cell lines expressing EF1-eGFP-ZNF346 and Tet-On-3G-eGFP-ZNF346 were generated from an A549 WT parental cell line purchased from ATCC (CCL185).

Techniques: Fluorescence, Microscopy, Activation Assay, Transfection, Control, Staining, Two Tailed Test, Expressing, Immunofluorescence

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