Journal: Regenerative biomaterials

Article Title: Fe-Zn alloy, a new biodegradable material capable of reducing ROS and inhibiting oxidative stress.

doi: 10.1093/rb/rbae002

Figure Lengend Snippet: Figure 5. Effects of different positions around alloy samples on ROS production, cell viability and DMA damage. (A) Schematic diagram of P1, P2 and P3 positions around the alloy (B–D) are ROS production, cell viability (living cell/green, dead cell/red) and DNA damage, respectively, at P1, P2 and P3 positions. DAPI (blue) was used for the identification of cell nuclei and c-H2AX fluorescence (green) for DNA damage.

Article Snippet: DNA damage assay kit (Beyotime Biotechnology, China) was selected to detect the DNA damage marker c-H2AX (phosphorylated H2AX) by immunofluorescence staining.

Techniques: Fluorescence

Journal: iScience

Article Title: SARS-CoV-2 nonstructural protein 1 suppresses host transcription by reducing RNA polymerase II levels

doi: 10.1016/j.isci.2025.114233

Figure Lengend Snippet: NSP1 protein impeded the gene expression of housekeeping genes (A) Analysis of mCherry expression in 293T cells co-transfected with mCherry plasmids and increasing amounts of Flag-NSP1 plasmids. Scale bars, 100 μm. (B) Analysis of GFP expression in 293T cells transfected with eGFP plasmids, with or without the SARS-CoV-2 leader sequence, in the presence of increasing amounts of Flag-NSP1 plasmids. Scale bars, 100 μm. (C) Schematic representation of Flag-NSP1 overexpression constructs. The SARS-CoV-2 leader sequence was inserted downstream of the promoter and upstream of the Flag-NSP1 coding sequence, followed by IRES-eGFP sequence. (D) Western blot analysis of GAPDH, eGFP, and Flag-NSP1 protein levels in H1299 cells transfected with Flag, Flag-NSP1, or Flag-NSP1-KH plasmids. Antibodies used are indicated on the right. (E) Immunofluorescence analysis of H1299 cells transfected with Flag-NSP1 or Flag-NSP1-KH plasmids. NSP1 proteins were detected using anti-Flag antibody (red), the nuclear envelope marker Lamin A/C (green), and DAPI (blue). Scale bars, 10 μm. (F and G) RT-qPCR analysis of ACTB and GAPDH mRNA levels in H1299 cells transfected with the indicated plasmids before (F) or after (G) FACS sorting for eGFP-positive cells. Expression levels were normalized to 18S rRNA and presented relative to Flag control (set as 1; n = 3 per group; mean ± SD; Student’s t test; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, no significance). (H) RT-qPCR analysis of ACTB and GAPDH mRNA levels in eGFP-positive and eGFP-negative H1299 cells transfected with the indicated plasmids. Expression levels were normalized to 18S rRNA and presented relative to Flag control in eGFP-negative cells (set as 1; n = 3 per group; mean ± SD; Student’s t test; ∗∗ p < 0.01; ns, no significance).

Article Snippet: We used following antibodies for Western blot, immunoprecipitation and immunofluorescence staining: β-actin (4970, CST), GADPH (2118, CST), GFP (ab290, Abcam), mCherry (ab183628, Abcam), NSP1 (ab284631, Abcam), Lamin A/C (4777, CST), Pol II (05-623, Sigma Aldrich), Flag (14793, CST), and Rabbit IgG (66362, CST).

Techniques: Gene Expression, Expressing, Transfection, Sequencing, Over Expression, Construct, Western Blot, Immunofluorescence, Marker, Quantitative RT-PCR, Control

Journal: MedComm

Article Title: Host Translational Control by Stress Granules Promotes Mycobacterium tuberculosis Pathogenesis

doi: 10.1002/mco2.70479

Figure Lengend Snippet: Mtb infection triggers robust SG formation in macrophages. (A) Immunofluorescence of lung tissues from UN and infected mice. G3bp1 fluorescence intensity was visualized in rainbow color to highlight differences in signal intensity. Scale bar indicates 50 µm. (B, C) Quantification of G3bp1 mean fluorescence intensity (MFI) (B) and G3bp1‐positive foci per area (C) from (A). Data are presented as mean ± SEM. **** p < 0.0001. N = 12. (D) Immunoblot of SG‐related proteins in lung lysates of UN and Mtb‐infected mice. (E) Immunofluorescence of SG markers in BMDMs infected with Mtb (MOI 1, 12 hpi). Scale bar indicates 10 µm. (F) Immunoblot of SG proteins in BMDMs at indicated times (MOI 1). (G) Quantification of relative band intensities from (F), normalized to β‐Actin. Data are presented as relative band intensities compared to UN. **** p < 0.0001. N = 3. (H) Immunofluorescence G3bp1 in H37Rv‐GFP infected BMDMs (MOI 1, 12 hpi). White arrowhead indicates H37Rv‐GFP‐negative cells. Scale bar indicates 10 µm. (I) Quantification of G3bp1‐positive cell ratio in H37Rv‐GFP − cells or H37Rv‐GFP + cells from (H). **** p < 0.0001. N = 20.

Article Snippet: The following primary antibodies were used for immunofluorescence analysis: mouse anti‐α‐Tubulin (CST, 3873), rabbit anti‐β‐Actin (CST, 4970), rabbit anti‐G3bp1 (abcam, ab181150), mouse anti‐Eif3η (SCT, sc‐137214), mouse anti‐G3bp1 (SCT, sc‐365338), rabbit anti‐G3bp2 (CST, 31799), rabbit anti‐Rack1 (abcam, ab62735), mouse anti‐Pabp1 (SCT, sc‐166381), mouse anti‐Puromycin (Millipore, MABE343), mouse anti‐Raptor (SCT, sc‐81537), rabbit anti‐mTOR (CST, 2983), rabbit anti‐Astrin (Proteintech, 14726‐1‐AP), and rabbit anti‐Ndufa12 (abcam, ab192617).

Techniques: Infection, Immunofluorescence, Fluorescence, Western Blot

Journal: MedComm

Article Title: Host Translational Control by Stress Granules Promotes Mycobacterium tuberculosis Pathogenesis

doi: 10.1002/mco2.70479

Figure Lengend Snippet: Intracellular ATP level regulates the dynamics of SGs which affect innate immune activity of macrophages. (A) Immunofluorescence analysis of SGs BMDMs treated with 2‐DG or glucose depletion for 24 h. Scale bar indicates 10 µm. (B) Immunofluorescence analysis of SGs in BMDMs infected with Mtb (MOI 1). Scale bar indicates 10 µm. (C) Intracellular ATP concentrations in BMDMs from (A) were plotted with SG‐positive ratio. The ATP concentration (17.5 nmol/mg protein) correlating with 50% of SG‐positive cells is indicated with dotted line and considered as SG‐inducing ATP concentration. (D) Intracellular ATP concentrations of BMDMs from (B) were plotted with SG‐positive ratio. The SG‐inducing ATP concentration (17.5 nmol/mg protein) from (C) is indicated with dotted line. (E) Intracellular ATP concentrations in zymosan, LPS, and PMA‐treated BMDMs. n.s., nonsignificant, * p < 0.05, **** p < 0.0001. N = 3. (F) Intracellular ATP concentrations in Mtb‐infected BMDMs, following cytochalasin D (Cyto D) treatment. n.s., nonsignificant, **** p < 0.0001. N = 3. (G) Immunoblot analysis of SG markers in BMDMs ± Cyto D after Mtb infection (MOI 1, 24 hpi). (H) Intracellular ATP concentrations in BMDMs treated with empty‐liposome or ATPsome before Mtb infection (MOI 1). n.s., nonsignificant, *** p < 0.001, **** p < 0.0001. N = 3. (I) Immunoblot analysis of SG markers in BMDMs treated as in (H). (J) Immunofluorescence analysis of SGs in GFP‐Mtb‐infected BMDMs ± ATPsome. Scale bar indicates 10 µm. (K) Quantification of Mtb area per cell in (J). * p < 0.05. N = 15. (L) Intracellular Mtb survival in BMDMs ± ATPsome. Data from three independent experiments are shown as mean ± SEM. **** p < 0.0001. N = 3. (M) BMDM viability (LDH assay) after ATPsome or liposome treatment ± infection (MOI 1) Relative to 0 hpi control (Liposome). n.s., nonsignificant; compared within the same time point. N = 3. (N, O) ROS (N; N = 3) and nitrite (O; N = 3) production was measured in media of BMDMs (MOI 1, 24 hpi) ± ATPsome. * p < 0.05. (P) Experimental scheme for pre‐ or post‐treatment of ATPsome. (Q) Immunoblot analysis of BMDMs pre‐ or post‐treated with ATPsome as in (P) (MOI 1, 24 hpi). (R) Intracellular Mtb survival in BMDMs pre/post‐ATPsome treatments (MOI 1). Data from three independent experiments are shown as mean ± SEM. n.s., nonsignificant, ** p < 0.01, *** p < 0.001, **** p < 0.0001. N = 9.

Article Snippet: The following primary antibodies were used for immunofluorescence analysis: mouse anti‐α‐Tubulin (CST, 3873), rabbit anti‐β‐Actin (CST, 4970), rabbit anti‐G3bp1 (abcam, ab181150), mouse anti‐Eif3η (SCT, sc‐137214), mouse anti‐G3bp1 (SCT, sc‐365338), rabbit anti‐G3bp2 (CST, 31799), rabbit anti‐Rack1 (abcam, ab62735), mouse anti‐Pabp1 (SCT, sc‐166381), mouse anti‐Puromycin (Millipore, MABE343), mouse anti‐Raptor (SCT, sc‐81537), rabbit anti‐mTOR (CST, 2983), rabbit anti‐Astrin (Proteintech, 14726‐1‐AP), and rabbit anti‐Ndufa12 (abcam, ab192617).

Techniques: Activity Assay, Immunofluorescence, Infection, Concentration Assay, Western Blot, Lactate Dehydrogenase Assay, Control

Journal: MedComm

Article Title: Host Translational Control by Stress Granules Promotes Mycobacterium tuberculosis Pathogenesis

doi: 10.1002/mco2.70479

Figure Lengend Snippet: SGs suppress host protein synthesis to support Mtb survival. (A) GO analysis of 97 proteins with log 2 ratio (24 hpi/12 hpi) < −1.0 from (Figure ); top five GO categories are shown, ranked by p value. (B) Ribopuromycylation assay of de novo protein synthesis in Mtb‐infected BMDMs (MOI 1). * p < 0.05, *** p < 0.001. N = 3. (C) Ribopuromycylation assay in ISRIB‐treated or untreated BMDMs, during Mtb infection (MOI 1). ** p < 0.01, *** p < 0.001. N = 3. (D) Puromycin immunofluorescence in infected BMDMs ± ISRIB (MOI 1, 12 hpi). Puromycin signal intensity was visualized using a rainbow color scale. Scale bar indicates 20 µm. (E) MFI of the puromycin from (D). Data are presented as mean ± SEM. ** p < 0.01. N = 10. (F) SG‐positive BMDM ratio with or without ISRIB (MOI 1, 12 hpi) were plotted as violin plot with median and quartiles. (G) Intracellular survival of Mtb in ISRIB‐treated or untreated BMDMs (MOI 1). Data are presented as mean ± SEM. n.s., nonsignificant, *** p < 0.001, compared within the same time point. N = 9. (H) Cell viability (LDH assay) of ISRIB‐treated or untreated BMDMs (MOI 1). Relative to 0 hpi ISRIB ‐. n.s., nonsignificant; compared within the same time point. N = 5. (I) Immunofluorescence of SGs in WT (siCont.) and G3bp1/2‐dKD (siG3bp1,2) BMDMs (MOI 1, 24 hpi). Scale bar indicates 20 µm. (J) Quantification of SG‐positive cells from (I). Both Eif3η‐ and Rack1‐positive puncta are considered as SGs. **** p < 0.0001, compared to siCont. N = 15. (K) Immunofluorescence of puromycin in WT and SG neg BMDMs (MOI 1, 12 hpi). Rainbow color gradient indicates puromycin signal intensity. Scale bar indicates 20 µm. (L) MFI of puromycin from (K). ** p < 0.01. N = 10. (M) Ribopuromycylation assay in Mtb‐infected SG neg BMDMs (MOI 1, 12 hpi). (N) Relative puromycin intensity from (M), normalized to β‐Actin. *** p < 0.001, **** p < 0.0001. N = 3. (O) Intracellular survival of Mtb in WT and SG neg BMDMs (MOI 1). Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001. N = 9. (P) Cell viability (LDH assay) of WT and SG neg BMDMs (MOI 1). Relative to 0 hpi siControl (siCont.). n.s., nonsignificant; compared within the same time point. N = 3.

Article Snippet: The following primary antibodies were used for immunofluorescence analysis: mouse anti‐α‐Tubulin (CST, 3873), rabbit anti‐β‐Actin (CST, 4970), rabbit anti‐G3bp1 (abcam, ab181150), mouse anti‐Eif3η (SCT, sc‐137214), mouse anti‐G3bp1 (SCT, sc‐365338), rabbit anti‐G3bp2 (CST, 31799), rabbit anti‐Rack1 (abcam, ab62735), mouse anti‐Pabp1 (SCT, sc‐166381), mouse anti‐Puromycin (Millipore, MABE343), mouse anti‐Raptor (SCT, sc‐81537), rabbit anti‐mTOR (CST, 2983), rabbit anti‐Astrin (Proteintech, 14726‐1‐AP), and rabbit anti‐Ndufa12 (abcam, ab192617).

Techniques: Infection, Immunofluorescence, Lactate Dehydrogenase Assay

Journal: MedComm

Article Title: Host Translational Control by Stress Granules Promotes Mycobacterium tuberculosis Pathogenesis

doi: 10.1002/mco2.70479

Figure Lengend Snippet: SGs sequester mTORC1 and inhibit cap‐dependent translation during infection. (A) Heatmap of TORC1 complex protein abundance in Mtb‐infected BMDMs (GO: 0031931; MGI v6.22). (B) Immunoblots of mTORC1 activity over time in infected BMDMs (MOI 1); bar graphs show phospho/total ratios. N = 3. (C) Immunoblots of cap‐dependent translation‐related proteins in infected BMDMs (MOI 1). (D) Heatmap of Spearman correlation coefficients ( ρ ) for pairwise expression patterns of proteins analyzed in (C). (E) Immunofluorescence showing colocalization of mTOR and Raptor with G3bp1 in infected macrophages (MOI 1, 12 hpi). Scale bar indicates 5 µm. (F) Dual‐color pixel analysis quantifying colocalization of mTORC1 components with G3bp1 from (E). Pearson correlation coefficients ( r ) are shown. (G) Co‐immunoprecipitation of G3bp1 with mTOR and Raptor from infected BMDMs (MOI 1, 24 hpi). (H) PLA showing spatial interaction of mTOR or Raptor with G3bp1 in infected BMDMs (MOI 1, 12 hpi). Scale bar indicates 5 µm. (I) Immunofluorescence of Astrin colocalizing with G3bp1 and Raptor in infected BMDMs (MOI 1, 12 hpi). Scale bar indicates 4 µm. (J) Immunoblot of Astrin expression in infected BMDMs (MOI 1). (K) PLA showing Astrin interactions with G3bp1 and Raptor in infected BMDMs (MOI 1, 12 hpi). Scale bar indicates 5 µm. (L) Immunoblots of SG markers, mTORC1, cap‐dependent translation proteins, and puromycin incorporation in SG neg BMDMs (MOI 1, 12 hpi).

Article Snippet: The following primary antibodies were used for immunofluorescence analysis: mouse anti‐α‐Tubulin (CST, 3873), rabbit anti‐β‐Actin (CST, 4970), rabbit anti‐G3bp1 (abcam, ab181150), mouse anti‐Eif3η (SCT, sc‐137214), mouse anti‐G3bp1 (SCT, sc‐365338), rabbit anti‐G3bp2 (CST, 31799), rabbit anti‐Rack1 (abcam, ab62735), mouse anti‐Pabp1 (SCT, sc‐166381), mouse anti‐Puromycin (Millipore, MABE343), mouse anti‐Raptor (SCT, sc‐81537), rabbit anti‐mTOR (CST, 2983), rabbit anti‐Astrin (Proteintech, 14726‐1‐AP), and rabbit anti‐Ndufa12 (abcam, ab192617).

Techniques: Infection, Quantitative Proteomics, Western Blot, Activity Assay, Expressing, Immunofluorescence, Immunoprecipitation

Journal: MedComm

Article Title: Host Translational Control by Stress Granules Promotes Mycobacterium tuberculosis Pathogenesis

doi: 10.1002/mco2.70479

Figure Lengend Snippet: SGs impair mitochondrial complex I activity during Mtb infection. (A) Scheme illustrating the breakdown of oxygen consumption following the addition of the specified inhibitors for assessing oxygen consumption rates (OCRs). (B) OCRs in WT and SG neg macrophages infected with Mtb (MOI 1). Cells were transfected with either siControl (left) or siG3bp1/2 (right) for 48 h before infection. (C–E) Quantification of relative basal (C), maximal (D), and ATP‐linked respirations (E) from (B). Normalized to each UN control (pink‐colored dotted line). Pink p value versus UN, black p value versus siCont. n.s., nonsignificant, * p < 0.05, ** p < 0.01. N = 3. (F) Mitochondrial membrane potential (JC‐1 red/green ratio) in infected WT and SG neg BMDMs (MOI 1; normalized to UN; pink dotted line). n.s., nonsignificant, **** p < 0.0001. N = 3. (G) Intracellular ATP concentrations in infected WT and SG neg BMDMs (MOI 1). **** p < 0.0001. N = 3. (H) Lactate secretion from UN or Mtb‐infected WT and SG neg BMDMs (MOI 1). n.s., nonsignificant, * p < 0.05, ** p < 0.01. N = 3. (I) STRING analysis (v11.5) of 37 proteins upregulated in SG neg BMDMs and simultaneously present in the SG proteome, grouped into translation (Cluster 1), mitochondria (Cluster 2), and nuclear import complex (Cluster 3). (J) Immunoblot of whole‐cell lysate (WCL), cytosolic fraction, and mitochondria fraction from Mtb‐infected WT and SG neg BMDMs (MOI 1, 12 hpi). (K) Immunofluorescence analysis of G3bp1‐Ndufa12 colocalization in Mtb‐infected WT and SG neg BMDMs (MOI 1, 12 hpi); line profiles show fluorescence intensity (white arrow). Scale bar indicates 5 µm. (L) Mitochondrial complex I activity in Mtb‐infected WT and SG neg BMDMs (MOI 1, 12 hpi); rotenone as positive control. Relative mitochondrial complex I activity is plotted, compared to uninfected siControl cells. n.s., nonsignificant, **** p < 0.0001. N = 3.

Article Snippet: The following primary antibodies were used for immunofluorescence analysis: mouse anti‐α‐Tubulin (CST, 3873), rabbit anti‐β‐Actin (CST, 4970), rabbit anti‐G3bp1 (abcam, ab181150), mouse anti‐Eif3η (SCT, sc‐137214), mouse anti‐G3bp1 (SCT, sc‐365338), rabbit anti‐G3bp2 (CST, 31799), rabbit anti‐Rack1 (abcam, ab62735), mouse anti‐Pabp1 (SCT, sc‐166381), mouse anti‐Puromycin (Millipore, MABE343), mouse anti‐Raptor (SCT, sc‐81537), rabbit anti‐mTOR (CST, 2983), rabbit anti‐Astrin (Proteintech, 14726‐1‐AP), and rabbit anti‐Ndufa12 (abcam, ab192617).

Techniques: Activity Assay, Infection, Transfection, Control, Membrane, Western Blot, Immunofluorescence, Fluorescence, Positive Control

Journal: MedComm

Article Title: Host Translational Control by Stress Granules Promotes Mycobacterium tuberculosis Pathogenesis

doi: 10.1002/mco2.70479

Figure Lengend Snippet: Inhibition of SGs restores macrophage immune function and restricts Mtb in vivo. (A) Experimental scheme for the in vivo experiments. Mice were transfected with siRNAs via intravenous (i.v.) injection, followed by intratracheal infection with Mtb for 7 days. Seven days postinfection, siRNA transfection was repeated, and the mice were sacrificed another 7 days later for experiments. (B) Immunofluorescence analysis of lung tissues from siRNA‐transfected uninfected (UN) or Mtb‐infected mice. G3bp1 fluorescence intensity was visualized in rainbow color. Scale bar indicates 20 µm. (C) Mean fluorescence intensity (MFI) of G3bp1 signals from (B). * p < 0.05. N = 10. (D) Immunoblot analysis of SG markers, mTORC1, and cap‐dependent translation proteins in lung lysates. (E) Quantification of relative band intensities from (D). Data are presented as relative band intensities compared to uninfected siControl group (UN/siCont.). * p < 0.05, *** p < 0.001. N = 3. (F) H&E staining of lung tissues from siRNA‐transfected uninfected (UN) or Mtb‐infected mice. Scale bar indicates 200 µm. (G) Quantification of TNF and MCP‐1 levels in mouse serum. Data are presented as mean ± SEM. N = 5. (H) Lung CFU in Mtb‐infected mouse lung tissues. Data are presented as mean ± SEM. **** p < 0.0001. N = 7. (I) Immunofluorescence analysis of lung tissues from ISRIB‐treated and Mtb‐infected mice. G3bp1 fluorescence intensity was visualized in rainbow color. Scale bar indicates 10 µm. (J) MFI of G3bp1 from (H). **** p < 0.0001. N = 10. (K) Lung CFU in ISRIB‐treated and Mtb‐infected mouse lung tissues. Data are presented as mean ± SEM. * p < 0.05. N = 9.

Article Snippet: The following primary antibodies were used for immunofluorescence analysis: mouse anti‐α‐Tubulin (CST, 3873), rabbit anti‐β‐Actin (CST, 4970), rabbit anti‐G3bp1 (abcam, ab181150), mouse anti‐Eif3η (SCT, sc‐137214), mouse anti‐G3bp1 (SCT, sc‐365338), rabbit anti‐G3bp2 (CST, 31799), rabbit anti‐Rack1 (abcam, ab62735), mouse anti‐Pabp1 (SCT, sc‐166381), mouse anti‐Puromycin (Millipore, MABE343), mouse anti‐Raptor (SCT, sc‐81537), rabbit anti‐mTOR (CST, 2983), rabbit anti‐Astrin (Proteintech, 14726‐1‐AP), and rabbit anti‐Ndufa12 (abcam, ab192617).

Techniques: Inhibition, In Vivo, Transfection, Injection, Infection, Immunofluorescence, Fluorescence, Western Blot, Staining