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Proteintech immunofluorescence analysis

a Schematic diagram of RNA-seq. Created in BioRender. Chen, P. (2026) https://BioRender.com/6rte857 . b Principal component analysis (PCA) of genes. c Heatmap showing differentially expressed genes in different groups; three biological replicates are shown. d Volcano plots of differentially expressed genes. P values were calculated using DESeq2 analysis with the Wald test. e Gene set enrichment analysis (GSEA) results of different groups. f Simultaneous imaging (left) and quantification (right) of cytosolic and mitochondrial Ca 2+ using Fluo-4/AM and Rhod-2/AM ( n = 3). g <t>Immunofluorescence</t> staining (left) and quantification (right) of CHOP and ATF4 protein levels ( n = 3). h Representative confocal image of human NPCs stained for calnexin (green, ER) and TOMM20 (red, mitochondria). Merged panel images were processed using Fiji/ImageJ (white dots indicate colocalized pixels). i Mander’s coefficients (M1) of ( h ). n = 3 biological replicates, 20 images were acquired and analyzed from each group. j Transmission electron microscopy (TEM) images of mitochondrial and ER morphology. The ER and mitochondria were graphically reconstructed: mitochondria (red); ER (green). k Quantification of the mitochondrial–ER contact length in ( j ). l Mitochondrial count per field from TEM in ( j ). m Quantification of ER lengths per field from ( j ). Measurements in ( k – m ) are from n = 3 biological replicates, with the normal group = 50 cells from 65 fields and the degeneration group = 50 cells from 63 fields. OMM: Outer mitochondrial membrane. n Calcein fluorescence (left) and quantification (right) of human NPCs ( n = 3). o Reactive oxygen species (ROS) staining (left) and quantification (right) of human NPCs ( n = 3). p JC-1 staining (left) and quantification (right) of human NPCs ( n = 3). All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using two-tailed Student’s t test ( f , g , i , and k – p ). Scale bars: 50 µm ( f – h , n – p ); 1 µm ( j ). Source data are provided as a Source Data file.

Immunofluorescence Analysis, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 198 article reviews

immunofluorescence analysis - by Bioz Stars, 2026-08

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1) Product Images from "Natural photosynthetic system for restoring homeostasis of animal organelle interaction network"

Article Title: Natural photosynthetic system for restoring homeostasis of animal organelle interaction network

Journal: Nature Communications

doi: 10.1038/s41467-026-69825-y

Figure Legend Snippet: a Schematic diagram of RNA-seq. Created in BioRender. Chen, P. (2026) https://BioRender.com/6rte857 . b Principal component analysis (PCA) of genes. c Heatmap showing differentially expressed genes in different groups; three biological replicates are shown. d Volcano plots of differentially expressed genes. P values were calculated using DESeq2 analysis with the Wald test. e Gene set enrichment analysis (GSEA) results of different groups. f Simultaneous imaging (left) and quantification (right) of cytosolic and mitochondrial Ca 2+ using Fluo-4/AM and Rhod-2/AM ( n = 3). g Immunofluorescence staining (left) and quantification (right) of CHOP and ATF4 protein levels ( n = 3). h Representative confocal image of human NPCs stained for calnexin (green, ER) and TOMM20 (red, mitochondria). Merged panel images were processed using Fiji/ImageJ (white dots indicate colocalized pixels). i Mander’s coefficients (M1) of ( h ). n = 3 biological replicates, 20 images were acquired and analyzed from each group. j Transmission electron microscopy (TEM) images of mitochondrial and ER morphology. The ER and mitochondria were graphically reconstructed: mitochondria (red); ER (green). k Quantification of the mitochondrial–ER contact length in ( j ). l Mitochondrial count per field from TEM in ( j ). m Quantification of ER lengths per field from ( j ). Measurements in ( k – m ) are from n = 3 biological replicates, with the normal group = 50 cells from 65 fields and the degeneration group = 50 cells from 63 fields. OMM: Outer mitochondrial membrane. n Calcein fluorescence (left) and quantification (right) of human NPCs ( n = 3). o Reactive oxygen species (ROS) staining (left) and quantification (right) of human NPCs ( n = 3). p JC-1 staining (left) and quantification (right) of human NPCs ( n = 3). All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using two-tailed Student’s t test ( f , g , i , and k – p ). Scale bars: 50 µm ( f – h , n – p ); 1 µm ( j ). Source data are provided as a Source Data file.

Techniques Used: RNA Sequencing, Imaging, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Membrane, Fluorescence, Two Tailed Test

a ATP levels of NPCs with different treatments ( n = 6). b The effects of NM-NTUs on mRNA expression levels of NPCs ( n = 3). c Immunofluorescence staining and quantification of protein levels in NPCs ( n = 3). d Heatmap of differentially expressed genes in NPCs; six biological replicates are shown. e PCA of genes in NPCs with different treatments. f Volcano plots of genes between the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU + dark group. P values were calculated using DESeq2 analysis with the Wald test. The Benjamini and Hochberg method was used for multiple comparison adjustments. g GSEA results of pathways in the IL-1β + NM-NTU + light group compared with those in the IL-1β + NM-NTU + dark group. All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using one-way ANOVA ( a – c ). Scale bar: 50 µm ( c ). Source data are provided as a Source Data file.

Figure Legend Snippet: a ATP levels of NPCs with different treatments ( n = 6). b The effects of NM-NTUs on mRNA expression levels of NPCs ( n = 3). c Immunofluorescence staining and quantification of protein levels in NPCs ( n = 3). d Heatmap of differentially expressed genes in NPCs; six biological replicates are shown. e PCA of genes in NPCs with different treatments. f Volcano plots of genes between the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU + dark group. P values were calculated using DESeq2 analysis with the Wald test. The Benjamini and Hochberg method was used for multiple comparison adjustments. g GSEA results of pathways in the IL-1β + NM-NTU + light group compared with those in the IL-1β + NM-NTU + dark group. All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using one-way ANOVA ( a – c ). Scale bar: 50 µm ( c ). Source data are provided as a Source Data file.

Techniques Used: Expressing, Immunofluorescence, Staining, Comparison

a Schematic diagram of the therapy. b , c Representative MRI images of rabbit IVD ( b ) and T2-weighted signal intensity of the NP in each group ( c ) ( n = 4). d Macroscopic view of IVD samples. The region highlighted by the red square is shown at higher magnification. The nucleus pulposus is delineated by a red dashed circle. e The H&E, Alcian blue, and Safranin O-Fast Green staining images of IVD samples. f Histological grade evaluation according to H&E staining at 4 and 8 weeks in each group ( n = 4). g Alcian blue staining quantification of rabbit IVDs at 4 and 8 weeks ( n = 4). h Safranin O-Fast Green staining quantification of rabbit IVDs at 4 and 8 weeks ( n = 4). i Immunofluorescence staining images of Col II and aggrecan of IVD samples. All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using one-way ANOVA ( c , g , h ) or nonparametric Kruskal–Wallis test ( f ). Scale bars: 400 µm ( e ); 200 μm ( i ). The regions of interest are marked by red arrows ( b ). Source data are provided as a Source Data file.

Figure Legend Snippet: a Schematic diagram of the therapy. b , c Representative MRI images of rabbit IVD ( b ) and T2-weighted signal intensity of the NP in each group ( c ) ( n = 4). d Macroscopic view of IVD samples. The region highlighted by the red square is shown at higher magnification. The nucleus pulposus is delineated by a red dashed circle. e The H&E, Alcian blue, and Safranin O-Fast Green staining images of IVD samples. f Histological grade evaluation according to H&E staining at 4 and 8 weeks in each group ( n = 4). g Alcian blue staining quantification of rabbit IVDs at 4 and 8 weeks ( n = 4). h Safranin O-Fast Green staining quantification of rabbit IVDs at 4 and 8 weeks ( n = 4). i Immunofluorescence staining images of Col II and aggrecan of IVD samples. All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using one-way ANOVA ( c , g , h ) or nonparametric Kruskal–Wallis test ( f ). Scale bars: 400 µm ( e ); 200 μm ( i ). The regions of interest are marked by red arrows ( b ). Source data are provided as a Source Data file.

Techniques Used: Staining, Immunofluorescence

Image Search Results

Journal: Nature Communications

Article Title: Natural photosynthetic system for restoring homeostasis of animal organelle interaction network

doi: 10.1038/s41467-026-69825-y

Figure Lengend Snippet: a Schematic diagram of RNA-seq. Created in BioRender. Chen, P. (2026) https://BioRender.com/6rte857 . b Principal component analysis (PCA) of genes. c Heatmap showing differentially expressed genes in different groups; three biological replicates are shown. d Volcano plots of differentially expressed genes. P values were calculated using DESeq2 analysis with the Wald test. e Gene set enrichment analysis (GSEA) results of different groups. f Simultaneous imaging (left) and quantification (right) of cytosolic and mitochondrial Ca 2+ using Fluo-4/AM and Rhod-2/AM ( n = 3). g Immunofluorescence staining (left) and quantification (right) of CHOP and ATF4 protein levels ( n = 3). h Representative confocal image of human NPCs stained for calnexin (green, ER) and TOMM20 (red, mitochondria). Merged panel images were processed using Fiji/ImageJ (white dots indicate colocalized pixels). i Mander’s coefficients (M1) of ( h ). n = 3 biological replicates, 20 images were acquired and analyzed from each group. j Transmission electron microscopy (TEM) images of mitochondrial and ER morphology. The ER and mitochondria were graphically reconstructed: mitochondria (red); ER (green). k Quantification of the mitochondrial–ER contact length in ( j ). l Mitochondrial count per field from TEM in ( j ). m Quantification of ER lengths per field from ( j ). Measurements in ( k – m ) are from n = 3 biological replicates, with the normal group = 50 cells from 65 fields and the degeneration group = 50 cells from 63 fields. OMM: Outer mitochondrial membrane. n Calcein fluorescence (left) and quantification (right) of human NPCs ( n = 3). o Reactive oxygen species (ROS) staining (left) and quantification (right) of human NPCs ( n = 3). p JC-1 staining (left) and quantification (right) of human NPCs ( n = 3). All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using two-tailed Student’s t test ( f , g , i , and k – p ). Scale bars: 50 µm ( f – h , n – p ); 1 µm ( j ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence analysis, the following antibodies were used: KRT19 (Proteintech, 10712-1-AP, 1:200), CD24 (Proteintech, 10600-1-AP, 1:200), CHOP (Proteintech, 15204-1-AP, 1:100); ATF4 (Proteintech, 60035-1-Ig, 1:200); Col II (Proteintech, 28459-1-AP, 1:800); aggrecan (Proteintech, 13880-1-AP, 1:200); MMP13 (Proteintech, 18165-1-AP, 1:200); ADAMTS-5 (Affinity Biosciences, DF13268, 1:200); TOMM20 (Abcam, ab56783, 1:200); Calnexin (Proteintech, 10427-2-AP, 1:200).

Techniques: RNA Sequencing, Imaging, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Membrane, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Natural photosynthetic system for restoring homeostasis of animal organelle interaction network

doi: 10.1038/s41467-026-69825-y

Figure Lengend Snippet: a ATP levels of NPCs with different treatments ( n = 6). b The effects of NM-NTUs on mRNA expression levels of NPCs ( n = 3). c Immunofluorescence staining and quantification of protein levels in NPCs ( n = 3). d Heatmap of differentially expressed genes in NPCs; six biological replicates are shown. e PCA of genes in NPCs with different treatments. f Volcano plots of genes between the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU + dark group. P values were calculated using DESeq2 analysis with the Wald test. The Benjamini and Hochberg method was used for multiple comparison adjustments. g GSEA results of pathways in the IL-1β + NM-NTU + light group compared with those in the IL-1β + NM-NTU + dark group. All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using one-way ANOVA ( a – c ). Scale bar: 50 µm ( c ). Source data are provided as a Source Data file.

Techniques: Expressing, Immunofluorescence, Staining, Comparison

Journal: Nature Communications

Article Title: Natural photosynthetic system for restoring homeostasis of animal organelle interaction network

doi: 10.1038/s41467-026-69825-y

Figure Lengend Snippet: a Schematic diagram of the therapy. b , c Representative MRI images of rabbit IVD ( b ) and T2-weighted signal intensity of the NP in each group ( c ) ( n = 4). d Macroscopic view of IVD samples. The region highlighted by the red square is shown at higher magnification. The nucleus pulposus is delineated by a red dashed circle. e The H&E, Alcian blue, and Safranin O-Fast Green staining images of IVD samples. f Histological grade evaluation according to H&E staining at 4 and 8 weeks in each group ( n = 4). g Alcian blue staining quantification of rabbit IVDs at 4 and 8 weeks ( n = 4). h Safranin O-Fast Green staining quantification of rabbit IVDs at 4 and 8 weeks ( n = 4). i Immunofluorescence staining images of Col II and aggrecan of IVD samples. All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using one-way ANOVA ( c , g , h ) or nonparametric Kruskal–Wallis test ( f ). Scale bars: 400 µm ( e ); 200 μm ( i ). The regions of interest are marked by red arrows ( b ). Source data are provided as a Source Data file.

Techniques: Staining, Immunofluorescence

Overexpression ACSL4 increases the therapeutic efficacy of anti-PD-1 antibody in vivo. (A) A schematic view of the treatment plan, (B) Images of isolated tumors from xenograft mice that received indicated treatment. Tumor volumes and weights in each group were calculated and displayed on the right. (C) Immunofluorescence staining of ferroptosis marker PTGS2 in isolated xenograft tumors with indicated treatment. Scale bar = 100 μm. Data represent the mean ± SEM of triplicates. P value was calculated by two-tailed Student’s t-test. ** P < 0.01, *** P < 0.001. n = 6

Journal: Discover Oncology

Article Title: Lipid metabolism-related genes correlate with immune microenvironment and regulate the efficacy of immunotherapy via ferroptosis in melanoma

doi: 10.1007/s12672-025-04163-x

Figure Lengend Snippet: Overexpression ACSL4 increases the therapeutic efficacy of anti-PD-1 antibody in vivo. (A) A schematic view of the treatment plan, (B) Images of isolated tumors from xenograft mice that received indicated treatment. Tumor volumes and weights in each group were calculated and displayed on the right. (C) Immunofluorescence staining of ferroptosis marker PTGS2 in isolated xenograft tumors with indicated treatment. Scale bar = 100 μm. Data represent the mean ± SEM of triplicates. P value was calculated by two-tailed Student’s t-test. ** P < 0.01, *** P < 0.001. n = 6

Article Snippet: The primary antibodies and dilutions for western blotting (WB) and immunofluorescence (IF) staining analysis are listed below: ACSL4 (22401-1-AP, Proteintech, Wuhan, China; 1:100 for IHC, 1:100 for IF and 1:5 000 for WB), ALOX5 (10021-1-Ig, Proteintech; 1:400 for IHC, and 1:500 for WB), PTGS2 (66351-1-Ig, Proteintech; 1:400 for IF), CD8α(ab22378,, Abcam; 1:300 for IF), ACTIN (66009-1-Ig, Proteintech; 1:10 000 for WB).

Techniques: Over Expression, Drug discovery, In Vivo, Isolation, Immunofluorescence, Staining, Marker, Two Tailed Test

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.

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

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.

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.

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

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.

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.

Techniques: Infection, Immunofluorescence, Lactate Dehydrogenase Assay

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).

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).

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

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

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.

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

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.

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.

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

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