sv40 enhancer sequence  (Addgene inc)

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: The eGFP-ZNF346 sequence was codon-optimized and synthesized by Twist Bioscience.

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

Journal: bioRxiv

Article Title: Macrophage adaptation to hypoxia in the tuberculous granuloma potentiates mycobacterium-induced mitochondrial damage and granuloma necrosis

doi: 10.64898/2026.02.06.702658

Figure Lengend Snippet: (A – C and G – K) Zebrafish larvae were infected with 100 – 150 WT or 280 ΔESX-1 Mm expressing BFP2, via the (A – C) hindbrain ventricle or (G - k caudal vein, 2 days post fertilization (dpf). (A) Time-lapse confocal micrographs of Mm-infected macrophages in an animal expressing Tg(mfap4:MTS-EGFP:myl7:RFP) cu72 and Tg(mpeg1:Brainbow) w201 2 dpi. Mm (blue), macrophage mitochondria (green), and macrophages (magenta) are shown. Arrowhead, fragmented mitochondria. Scale bar, 10 μm. (B) Confocal micrograph of granulomas in Tg(mfap4:MTS-EGFP) animals infected with WT or ΔESX-1 2 dpi, showing mitochondrial morphology in macrophages inside and outside of the granuloma. Macrophage mitochondria (green) and Mm (blue). Zoomed-in boxed regions highlight differences in mitochondrial morphology between macrophages outside (blue boxes) and inside (yellow boxes) the granuloma. The lower panels show 3D renderings of the mitochondrial fluorescence signal. Scale bar, 10 μm. (C) Quantification of mitochondrial sphericity in macrophages inside and outside granulomas in Tg(mfap4:MTS-EGFP) animals 2 dpi. Horizontal lines indicate pooled mean values. Error bars represent 95% confidence intervals. (D and E) Zebrafish larvae were infected with ∼400 WT or ∼800 ΔesxA Mm expressing tdTomato via the hindbrain ventricle 2 dpf. Quantification of average mitochondrial sphericity in macrophages of Tg(mfap4:MTS-EGFP) animals infected with (D) WT and ΔesxA Mm, and (E) ΔesxA Mm or ΔexsA Mm complemented with WT or M83I point mutant Mtb esxA . Horizontal lines indicate pooled mean values. Error bars represent 95% confidence intervals. (F) Illustration of the FRET reporter to indicate caspases 3/7 activation. mNeonGreen (FRET donor) and mScarlet-I (FRET acceptor) fluorescent proteins are covalently linked with a peptide containing the DEVD caspase 3/7 recognition sequence. Intramolecular FRET is lost in the presence of active caspase-3 or −7. (G) Time-lapse confocal micrographs of Mm-infected macrophages in Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) showing macrophage death with (top) and without (bottom) caspase 3/7 activation 2 days post infection (dpf). Mm (blue) and macrophages (magenta or green) are shown. Scale bar, 10 μm. (H) Quantification of macrophage death modes in wild-type animals expressing Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) and infected with BFP2-expressing WT Mm 2 dpi. (I) Quantification of macrophage deaths at 2 dpi associated with FRET loss in animals infected intravenously with BFP2-expressing WT Mm and treated with QVD-OPH (50 μM) or 0.5% DMSO immediately following infection. (J) Quantification of observed macrophage death in wild-type animals expressing Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) and infected with WT or ΔESX-1 Mm 2 dpi. (K) Quantification of macrophage death modes in wild-type animals expressing Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) and infected with BFP2-expressing ΔESX-1 Mm 2 dpi. (L) Time-lapse confocal micrographs of a dying infected macrophage being phagocytosed by nearby macrophages in an Tg(mfap4: mNeonGreen-DEVD-mScarlet-i) animal 2 dpi. Mm (blue), live macrophage (magenta), dying macrophage with caspase 3/7 activation (green). Arrows and solid polygons, the dying macrophage before (magenta) and after (green) caspase 3/7 activation. Arrowheads and dashed polygons, uninfected (white), being infected (white and blue), and infected (blue) macrophages that phagocytose the dying macrophages. Scale bar, 20 μm. See Movie S1. Statistical significance was determined by (C and E) one-way ANOVA with Tukey post-test, (D and I) two-tailed, unpaired t-test, and (J) Fisher’s exact test. * p < 0.05 , ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 . (A – D, F – K) (A and G – K), Data are representative of two or more independent experiments.

Article Snippet: The plasmid used to generate the Tg(mfap4:MTS-EGFP:myl7:RFP) cu72 was created by cloning EGFP tagged with a mitochondrial targeting sequence (MTS-EGFP) from (RRID:Addgene_31241) into a pCS2-based Tol2 vector containing the mfap4 macrophage-specific promoter sequence and the myl7 promoter sequence driving RFP expression in the heart , using the Gibson Cloning kit (New England Biolabs).

Techniques: Infection, Expressing, Fluorescence, Mutagenesis, Activation Assay, Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Macrophage adaptation to hypoxia in the tuberculous granuloma potentiates mycobacterium-induced mitochondrial damage and granuloma necrosis

doi: 10.64898/2026.02.06.702658

Figure Lengend Snippet: 2 dpf zebrafish larvae were infected with 100 – 150 WT or 250 – 300 ΔESX-1 BFP2-expressing Mm and then treated with rapamycin (1 μM) or 0.5% DMSO (vehicle control) for the duration of the experiment. (A) Confocal micrographs of granulomas in Tg(mfap4:MTS-EGFP) animals 2 dpi, showing mitochondrial fragmentation in the rapamycin treated, WT Mm infected group. Mm (magenta) and macrophage mitochondria (green) are shown. Zoomed-in boxed regions show 3D renderings of the mitochondrial fluorescence signal. Scale bar, 10 μm. (B) Quantification of average mitochondrial sphericity in Tg(mfap4:MTS-EGFP) animals 2 dpi. Horizontal lines indicate pooled mean values. Error bars represent 95% confidence intervals. (C) Quantification of macrophage deaths through 6-hour timelapse imaging in Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) animals infected with WT Mm 3 dpi. (D) Quantification of macrophage death with or without caspases 3/7 activation through 6-hour timelapse imaging in Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) animals infected with WT Mm 3 dpi. (E) Time-lapse confocal micrographs of apoptotic WT Mm-infected macrophages undergoing efferocytosis (top) or secondary necrosis (bottom) in a Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) animal 3 dpi. Apoptotic macrophage before (magenta arrow) and after (green arrow) caspase-3/7 activation, efferocytic macrophage (white arrowhead). Scale bar, 20 μm. (F) Quantification of macrophage death followed by efferocytosis or secondary necrosis in Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) animals 3 dpi. (G) Quantification of the time interval between an infected macrophage dying and being re-phagocytosed in Tg(mfap4:mNeonGreen-DEVD-mScarlet-i) animals 3 dpi. Symbols represent individual Mm-infected, dying macrophages. Horizontal lines indicate mean values. Statistical significance was determined by (B) one-way ANOVA with Tukey post-test, (C, D, and F) Fisher’s exact test, and (G) two-tailed, unpaired t-test. ns p > 0.05, ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 . Data are representative of two independent experiments.

Article Snippet: The plasmid used to generate the Tg(mfap4:MTS-EGFP:myl7:RFP) cu72 was created by cloning EGFP tagged with a mitochondrial targeting sequence (MTS-EGFP) from (RRID:Addgene_31241) into a pCS2-based Tol2 vector containing the mfap4 macrophage-specific promoter sequence and the myl7 promoter sequence driving RFP expression in the heart , using the Gibson Cloning kit (New England Biolabs).

Techniques: Infection, Expressing, Control, Fluorescence, Imaging, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Macrophage adaptation to hypoxia in the tuberculous granuloma potentiates mycobacterium-induced mitochondrial damage and granuloma necrosis

doi: 10.64898/2026.02.06.702658

Figure Lengend Snippet: (A) Confocal micrograph of a granuloma with central necrosis isolated 4 weeks post infection (wpi) from a Tg(4xhre-tata:mCherry,cmlc2:eGFP) adult animal infected intraperitoneally with mWasabi-expressing Mm. Infected cells in non-necrotic regions of the granuloma (arrowheads) show low HIF activity compared to cells within the necrotic core (dashed lines). Hoechst-labelled nuclei (blue), Mm (green), HRE-driven mCherry expression (magenta). Scale bar, 10 μm. See Movie S2. (B) Overlaid widefield and bright field micrographs of vhl sa40757/sa40757 and wild-type siblings expressing Tg(4xhre-tata:mCherry,cmlc2:EGFP) 5 dpf. HRE-driven mCherry expression (red), constitutive epicardial GFP signal confirms transgene expression (blue). Scale bar, 1000 μm. Zebrafish were infected via the caudal vein (C – H, J, M and N) or the hindbrain ventricle (L) with tdTomato-expressing Mm of the indicated strains 2 dpf. (C) Confocal micrographs of WT (top) and vhl sa40757/sa40757 (bottom) Tg(mpeg1:YFP) animals 5 dpi. Scale bar, 50 μm. (D) Quantification of extracellular mycobacterial growth (cording) in animals from vhl sa40757/+ incross 5dpi. (E) Quantification of macrophages in Mm-infected and age-matched uninfected animals from vhl sa40757/+ incross expressing Tg(mpeg1:YFP) 5 dpi. Symbols represent individual animals. Horizontal lines indicate mean values. (F) Widefield micrograph of Mm fluorescence, in vhl sa40757/sa40757 and WT sibling 5dpi. Scale bar, 1000 μm. (G) Quantification of Mm fluorescent pixel counts (FPC) in animals from vhl sa40757/+ incross 5dpi. Symbols represent individual animals. Horizontal lines indicate mean values. (H) Quantification of mycobacterial cording 5 dpi. (I) Overlaid widefield and bright field micrographs of Tg(4xhre-tata:mCherry,cmlc2:EGFP) animals 5 dpf, three days after treatment with roxadustat (ROXA, 60 μM) or 0.5% DMSO. Scale bar 1000 μm. (J) Quantification of Mm cording in roxadustat (ROXA)- and vehicle-treated animals 5dpi. (K) Quantification of MitoTracker CMH 2 -Xros mean fluorescence intensity (MFI) in uninfected (bystander) and Mm-infected macrophages from Tg(mpeg1:YFP) animals treated with roxadustat (ROXA) or vehicle, 24 hours-post caudal vein infection with BFP2-expressing Mm and drug treatment. Symbols indicate values from individual macrophages. Horizontal lines indicate mean values. (L) Quantification of average mitochondrial sphericity in granuloma macrophages from WT and vhl G 0 crispants expressing Tg(mfap4:MTS-EGFP) 2 dpi. Horizontal lines indicate pooled mean values. Error bars represent 95% confidence intervals. (M) Quantification of Mm cording in WT animals infected with WT or Δ ESX-1 Mm and treated with roxadustat (ROXA) or vehicle 5dpi. (N) Mycobacterial cording in roxadustat (ROXA)- and vehicle-treated animals infected with ΔesxA Mm or ΔexsA Mm complemented with WT or point mutant Mtb esxA , 5dpi. Statistical significance was determined by (D, H, J, M, and N) Fisher exact test, and (E, G, K, and L) one-way ANOVA with Tukey post-test. ns p > 0.05 , * p < 0.05 , *** p < 0.001 , and **** p < 0.0001 . Data are representative of two (H and L) or three (D, G, J, K and M) independent experiments.

Article Snippet: The plasmid used to generate the Tg(mfap4:MTS-EGFP:myl7:RFP) cu72 was created by cloning EGFP tagged with a mitochondrial targeting sequence (MTS-EGFP) from (RRID:Addgene_31241) into a pCS2-based Tol2 vector containing the mfap4 macrophage-specific promoter sequence and the myl7 promoter sequence driving RFP expression in the heart , using the Gibson Cloning kit (New England Biolabs).

Techniques: Isolation, Infection, Expressing, Activity Assay, Fluorescence, Mutagenesis